Name: Optimized Analysis of Proteins from Xenopus Oocytes and Embryos by Immunoblotting (Video) | JoVE Journal
Uploaded: 2025-09-19T14:00:00.000+00:00
Duration: 9 min 32 s
Description: Learn immunoblotting techniques for protein analysis in Xenopus oocytes and embryos. This protocol enables detection of translational regulators and sulfate regulatory proteins.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

We thank Laura Vanderploeg for preparing figures and the Bill Bement lab for providing Xenopus oocytes. We also thank Marko Horb, Kelsey Coppenrath, and the National Xenopus Resource, Marine Biological Laboratory, Woods Hole, MA, for providing Xenopus tropicalis embryos. We would also like to thank Emily T. Johnson for her insightful comments on the manuscript. This work benefited from the support of Xenbase (http://www.xenbase.org/; RRID: SCR_003280). Work in the Sheets lab is supported by the NICHD (R01HD091921) and NIGMS (R01GM152615). Charlotte Kanzler is supported by the Biotechnology Training Program through the National Institute of General Medical Sciences of the National Institutes of Health (T32GM135066).&#160;

All work with animals (X. laevis) represented in this protocol is in accordance with and has been approved by the UW-Madison Institutional Animal Care and Use Committee. Details of equipment, reagents used, and antibody concentrations employed are listed in the Table of Materials. A selection of equipment can be viewed in Figure 2, below.
<img alt="Protein purification process using chromatography; includes electrophoresis setup and gel tray." class="xfigimg" src="/files/ftp_upload/69139/69139fig2.jpg" data-altupdatedat="1783396841000" data-alt="Figure 2"> 
Figure 2: Processed embryo extract and equipment for immunoblotting. (A) Centrifuged X. laevis stage VI oocyte extract. Lipids and fat-soluble proteins rise to the top while vitellogenin (yolk) and pigmented debris form a pellet. (B) Equipment for SDS-PAGE to separate proteins by molecular weight. (i) Power supply, (ii) Vertical electrophoresis tank, (iii) Electrophoresis tank lid, (iv) Electrode assembly, (v) Buffer dam, (vi) Precast electrophoresis gel; note the green comb (top) and blue sticker (bottom), each of which must be removed. (C) Equipment for membrane transfer. The vertical electrophoresis tank and lid are reused for transfer after a thorough rinse. (i) Roller to remove bubbles, (ii) Transfer clip, (iii) Small stir bar, (iv) Nitrocellulose membrane in between two protective blue sheets of paper, (v) Cellulose chromatography filter paper, (vi) Transfer sponge cushions, (vii) Glass baking dish for transfer assembly, (viii) Ice pack, (ix) Transfer core. (D) Other equipment. (i) Gel releaser (for trimming gel wells pre-transfer), (ii) Gel cassette opening lever, (iii) Forceps (for handling membrane), (iv) Container (for holding membrane), (v) container lid. <a href="https://www.jove.com/files/ftp_upload/69139/69139fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Collection of Xenopus oocytes and embryos
<ol>
	<li>Oocyte preparation
	<ol>
		<li>Obtain defolliculated X. laevis oocytes from a commercial supplier (Ecocyte Bio Science, Austin, Texas) or isolate and defolliculate as described8.</li>
		<li>Culture isolated oocytes in Modified Barth's Solution (MBS; 88 mM NaCl, 1 mM KCl, 0.4 mM CaCl2, 0.33 mM Ca(NO3)2, 0.8 mM MgSO4, 5 mM Tris-HCl, 2.4 mM NaHCO3, pH 7.4) until use.</li>
	</ol>
	</li>
	<li>Embryo preparation
	<ol>
		<li>Isolate X. laevis eggs and fertilize them as described9,10.</li>
		<li>Culture fertilized embryos in 0.25x Marc's Modified Ringer's solution11 (MMR) (1x MMR: 0.1 M NaCl, 2.0 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 5 mM HEPES).</li>
		<li>Prepare a solution of 2% cysteine in 0.1x MMR with the pH adjusted to 8.2 using NaOH.</li>
		<li>Remove 0.25x MMR from the dish used for fertilization and cover the embryos with the cysteine solution to remove the jelly coat12. Mix the solution gently so embryos are evenly exposed.</li>
		<li>Monitor embryos closely with a stereomicroscope from 2x to 25x magnification. After the jelly coat has dissolved (typically 3-5 min), the embryos will pack together closely. Avoid prolonged exposure to cysteine.</li>
		<li>Remove the cysteine solution and rinse embryos multiple times with 0.25x MMR.</li>
		<li>Culture in 0.25x MMR until the desired growth stage.</li>
	</ol>
	</li>
	<li>Collect the desired number of embryos or oocytes (recommended at least five per sample) in a 1.5 mL tube.</li>
	<li>Remove excess media with a pipettor, avoiding damaging the cells.</li>
	<li>Use samples immediately or store at -80 °C until use. Oocytes and embryos can be stored for several years.</li>
</ol>
2. Preparation of Xenopus extract
<ol>
	<li>Thaw samples on ice if frozen.</li>
	<li>Add 10 µL of chilled 10x cell lysis buffer diluted to 1x with double deionized water (see Table of Materials) per embryo/oocyte and homogenize samples with a micropestle on ice.</li>
	<li>Spin samples at 5000 g at 4 °C in a benchtop centrifuge for 10 min to pellet debris, including yolk and insoluble pigments.</li>
	<li>Remove supernatant to a separate 1.5 mL tube, taking care to avoid the pellet (Figure 2A).</li>
	<li>Add an equal volume of 2x Laemmli sample buffer supplemented with 5% w/v 2-mercaptoethanol to the supernatant and boil for 10 min at 95-100 °C to denature sample proteins.</li>
	<li>Use samples immediately or store at -20 °C for months to years.</li>
</ol>
3. SDS-PAGE
<ol>
	<li>Prepare SDS running buffer: 1x Tris-MOPS-SDS buffer (6.06 g of Tris base, 10.46 of g 3- (N-morpholino)propanesulfonic acid [MOPS], 1.0 g of SDS, 0.3 g of ethylenediaminetetraacetic acid [EDTA], double deionized water to 1000 mL).</li>
	<li>Remove a 4-12% bis-tris SDS precast gel from the packaging. Remove the sticker at the bottom of the gel.</li>
	<li>Insert the gel into the electrode assembly opposite a buffer dam. Place the electrode assembly into the vertical electrophoresis tank (Figure 2B).</li>
	<li>Fill the electrode assembly and the bottom of the electrophoresis tank with 1x Tris-MOPS-SDS running buffer.</li>
	<li>Gently remove the gel comb from the gel and pipette running buffer into the wells to remove bubbles and equilibrate the buffer.</li>
	<li>Carefully load 5 µL of pre-stained protein standards into the first well and 10 µL of each sample into the additional wells.</li>
	<li>(OPTIONAL) Load remaining wells with 10 µL of 1x Laemmli sample buffer to make the gel run more evenly.</li>
	<li>Place the lid on the electrophoresis tank and connect it to a power supply. Apply 200 V.</li>
	<li>Stop electrophoresis when the sample buffer dye front exits the gel.</li>
</ol>
4. Wet transfer of proteins onto a membrane
<ol>
	<li>While samples are electrophoresing, prepare 1 L of transfer buffer (3.0 g of Tris Base, 4.08 g of bicine, 900 mL of double deionized water, 100 mL of methanol). Pre-chill the transfer buffer at 4 °C. 
	CAUTION: Handle concentrated methanol stocks in a fume hood and avoid contact with skin. Methanol can be substituted with ethanol.</li>
	<li>Before the electrophoresis is complete, begin to assemble the transfer "sandwich" in the transfer clip (Figure 2C).
	<ol>
		<li>Fill a glass baking dish with chilled transfer buffer. In subsequent sandwich assembly steps, make sure materials are submerged in this buffer.</li>
		<li>Place the transfer clip in the dish with the black half lying against the bottom of the dish, the white half open and pointed upwards, and the transfer clip open to the left.</li>
		<li>Lay one or two fiber sponges flat on the black half of the transfer clip.</li>
		<li>Cut two 0.35 mm cellulose chromatography papers (filter papers) to size and lay one on top of the sponges.</li>
	</ol>
	</li>
	<li>After electrophoresis is complete, remove the gel from the electrophoresis tank.</li>
	<li>Carefully pry apart the plates of the gel with the gel cassette opening lever, ensuring that the gel remains attached to one of the plates (Figure 2D). Trim the wells from the top of the gel with the gel releaser.</li>
	<li>Place the gel, still attached to one half of the plastic cassette, onto the filter paper. Remove the cassette half so the gel remains on the filter paper.</li>
	<li>Cut a square of 0.45 µm nitrocellulose membrane to fit the dimensions of the gel. Remove the membrane from the protective paper, wet it briefly in transfer buffer, and place it on the gel. 
	NOTE: Handle the membrane carefully by the corners with gloves or forceps to avoid unwanted protein contamination of the membrane.</li>
	<li>Place a second filter paper on top of the nitrocellulose membrane. Gently but firmly remove any bubbles using a roller on top of the filter paper.</li>
	<li>Stack one to two more fiber sponges on top of the filter paper.</li>
	<li>Close the transfer cassette and clip it tightly shut, completing the 'sandwich'.</li>
	<li>Insert the transfer sandwich into the transfer core with the transfer clip facing upwards and the black side of the transfer clip facing the black side of the core.</li>
	<li>Place the transfer core into the electrophoresis tank. Add an ice pack and a stir bar to the tank so that the stir bar can rotate freely. 
	NOTE: In addition to or in place of an ice pack, the transfer can be run in a cold room at 4 °C in step 4.13.</li>
	<li>Fill the tank with chilled transfer buffer. Secure the lid firmly on top.</li>
	<li>Connect the transfer apparatus to the power supply, making sure to align electrode colors. Run the transfer for 1 h, 100 V, at 4 °C, with stir bar rotating.</li>
</ol>
5. Post-transfer membrane processing
<ol>
	<li>Prepare 10x Tris-Buffered Saline (100 mM Tris base; 1.5 M NaCl). Adjust pH to 8 and filter sterilize.</li>
	<li>Prepare 1x Tris-Buffered Saline with Tween-20 (TBSTw) by diluting 10x TBS solution 1:10 in 500 mL double deionized water and adding 500 µL Tween-20.</li>
	<li>Prepare blocking buffer (500 mL TBSTw, 25 g of nonfat milk powder). 
	NOTE: Nonfat milk can be substituted with 2-5% bovine serum albumin (BSA) in the blocking buffer. BSA should be used if the experimental application involves visualizing biotinylated proteins, as milk contains biotin.</li>
	<li>Once the transfer is complete, carefully remove and disassemble the sandwich and remove the nitrocellulose membrane. Mark which side of the membrane was contacting the gel, and keep this side face up in subsequent steps.</li>
	<li>Prepare 50 mL of Ponceau stain (0.5% Ponceau S, 1% acetic acid), a reversible stain for protein visualization. 
	CAUTION: Ponceau stain is corrosive. Avoid contact with skin.</li>
	<li>Place the membrane in a small container (as shown in Figure 2D) so that it lies flush against the bottom of the container. Cover the membrane in Ponceau stain to immersion and rock gently on a rocker for 5-10 min.</li>
	<li>Pour off the Ponceau stain. 
	NOTE: Ponceau stain can be reused multiple times.</li>
	<li>Rinse the membrane in repeated washes of double-deionized water until excess Ponceau is removed and protein bands in the lanes begin to resolve.</li>
	<li>Confirm the efficiency of the transfer by the presence of straight, even lanes with clearly resolved bands.</li>
	<li>Pour off any remaining liquid and immerse the blot in blocking buffer to prevent nonspecific antibody binding to the membrane in downstream steps.</li>
	<li>Rock gently on a rocker for 30-60 min at room temperature.</li>
</ol>
6. Antibody incubation
<ol>
	<li>Dilute the primary antibody in 10 mL of fresh blocking buffer to the desired concentration. 
	NOTE: The optimal concentration will have to be determined empirically for each new antibody. A typical starting point is 1:1000, or else follow the manufacturer's recommendations.</li>
	<li>Remove the blocking buffer and replace it with the antibody mixture. Incubate, rocking gently, overnight (~16 h) at 4 °C.</li>
	<li>Pour off the primary antibody solution and rinse the membrane with TBSTw by immersing it in the solution and quickly pouring it off. 
	NOTE: Most antibody mixtures can be saved and reused two to three times. This must be determined empirically for each antibody.</li>
	<li>Wash the membrane by immersing it in TBSTw and rocking gently at room temperature for 5 min. Perform three total 5 min washes.</li>
	<li>Dilute the secondary antibody in 30 mL of TBSTw to the desired concentration. 
	NOTE: Concentration will have to be determined empirically for each antibody. We typically use 1:30,000.</li>
	<li>Pour off the TBSTw wash solution and replace it with the secondary antibody mixture. Incubate, rocking gently, for 45 min at room temperature.</li>
	<li>Quickly rinse the membrane once with TBSTw followed by three 5-min TBSTw washes.</li>
</ol>
7. Imaging the membrane on a film developer
<ol>
	<li>In a darkroom, turn on the film developer and allow it to warm up.</li>
	<li>Cut two squares of plastic wrap large enough to encompass the membrane. Using forceps, place the nitrocellulose membrane' face up' on the first square of plastic wrap.</li>
	<li>In a 1.5 mL tube, mix equal amounts of the enhanced chemiluminescence (ECL) Luminol and Enhancer reagents. Using 1 mL of the mixed solution should cover most membranes.</li>
	<li>Pipette the ECL reagent onto the membrane and manipulate the membrane gently using the plastic wrap to ensure full coverage. Incubate for 1 min.</li>
	<li>Remove excess ECL reagent by grasping the membrane with forceps and gently shaking off liquid or wicking away liquid by touching a corner of the membrane to a paper towel.</li>
	<li>Place the membrane face up on the second square of plastic wrap and fold the plastic wrap over the membrane until it is loosely sealed. Tape the wrapped membrane securely into an autoradiography cassette.</li>
	<li>In the darkroom, place a film within the cassette, covering the taped membrane. Close the cassette securely and expose the film to the membrane for 1 min.</li>
	<li>Carefully remove the film, being careful not to drag the exposed film along the membrane. Feed it into the film developer.</li>
	<li>Upon evaluating the developed film, adjust exposure time with subsequent films until the desired protein visualization is achieved. If the protein visualization provides a weak signal, repeat the ECL reagent incubation step with a more sensitive ECL reagent and re-image.</li>
	<li>Align the developed film with the glow-in-the-dark markers and use this reference to mark the position of the pre-stained protein standards on the membrane onto the film.</li>
	<li>Once the imaging is complete, carefully remove the membrane from the plastic wrap and immerse it in TBSTw to wash away the remaining ECL reagent.</li>
</ol>
8. (Optional) Additional antibody incubations
NOTE: Stripping an immunoblot refers to the removal of the initial primary and secondary antibodies with a denaturing solution (stripping buffer) so the blot can be re-probed with a different antibody. Re-probing allows the same immunoblot to be analyzed for expression of different protein targets and to compare unknown proteins to well-characterized proteins that function as standards. Probe with the antibody that produces the comparatively weakest signal first. Doing so prevents artifacts caused by incompletely stripped antibodies in subsequent exposures and minimizes the impact of protein loss from repeated stripping of a blot.
<ol>
	<li>Strip the bound antibodies by immersing the membrane in stripping buffer and rocking gently for 5-10 min.</li>
	<li>Remove membrane from the stripping buffer and rinse briefly in TBSTw to remove any residual stripping solution.</li>
	<li>Immerse the membrane in blocking buffer and rock gently for 30 min.</li>
	<li>Make a new solution of primary antibody in blocking buffer with the desired concentration.</li>
	<li>Add the new primary antibody dilution to the membrane and incubate rocking gently overnight at 4 °C.</li>
	<li>Proceed from step 6.3 onwards. The membrane can be stripped and re-probed up to three additional times.</li>
</ol>

<ol><li>Gurdon, J. B., Wickens, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+Xenopus+oocytes+for+the+expression+of+cloned+genes%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">The use of Xenopus oocytes for the expression of cloned genes.</a> Methods Enzymol. 101, 370-386 (1983).</li>
<li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microinjection+of+Xenopus+embryos%5BTitle%5D)+AND+%22Cold+Spring+Harb+Protoc%22%5BJournal%5D)">Microinjection of Xenopus embryos.</a> Cold Spring Harb Protoc. 2010 (12), (2010).</li>
<li>Coller, J., Wickens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tethered+function+assays%3A+an+adaptable+approach+to+study+RNA+regulatory+proteins%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Tethered function assays: an adaptable approach to study RNA regulatory proteins.</a> Methods Enzymol. 429, 299-321 (2007).</li>
<li>Minshall, N., Allison, R., Marnef, A., Wilczynska, A., Standart, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Translational+control+assessed+using+the+tethered+function+assay+in+Xenopus+oocytes%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Translational control assessed using the tethered function assay in Xenopus oocytes.</a> Methods. 51 (1), 165-169 (2010).</li>
<li>Zhang, Y., Cooke, A., Park, S., Dewey, C. N., Wickens, M., Sheets, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bicaudal-C+spatially+controls+translation+of+vertebrate+maternal+mRNAs%5BTitle%5D)+AND+%22RNA%22%5BJournal%5D)">Bicaudal-C spatially controls translation of vertebrate maternal mRNAs.</a> RNA. 19 (11), 1575-1582 (2013).</li>
<li>Waghray, S., Williams, C., Coon, J. J., Wickens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Xenopus+CAF1+requires+NOT1-mediated+interaction+with+4E-T+to+repress+translation+in+vivo%5BTitle%5D)+AND+%22RNA%22%5BJournal%5D)">Xenopus CAF1 requires NOT1-mediated interaction with 4E-T to repress translation in vivo.</a> RNA. 21 (7), 1335-1345 (2015).</li>
<li>Neff, A. W., Wakahara, M., Jurand, A., Malacinski, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+analyses+of+cytoplasmic+rearrangements+which+follow+fertilization+and+accompany+symmetrization+of+inverted+Xenopus+eggs%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Experimental analyses of cytoplasmic rearrangements which follow fertilization and accompany symmetrization of inverted Xenopus eggs.</a> Development. 80 (1), 197-224 (1984).</li>
<li>Newman, K., Aguero, T., King, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+Xenopus+oocytes%5BTitle%5D)+AND+%22Cold+Spring+Harb+Protoc%22%5BJournal%5D)">Isolation of Xenopus oocytes.</a> Cold Spring Harb Protoc. 2018 (2), (2018).</li>
<li>Shaidani, N. -I., McNamara, S., Wlizla, M., Horb, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Obtaining+Xenopus+laevis+eggs%5BTitle%5D)+AND+%22Cold+Spring+Harb+Protoc%22%5BJournal%5D)">Obtaining Xenopus laevis eggs.</a> Cold Spring Harb Protoc. 2021 (3), (2021).</li>
<li>Shaidani, N. -I., McNamara, S., Wlizla, M., Horb, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Obtaining+Xenopus+laevis+embryos%5BTitle%5D)+AND+%22Cold+Spring+Harb+Protoc%22%5BJournal%5D)">Obtaining Xenopus laevis embryos.</a> Cold Spring Harb Protoc. 2021 (3), (2021).</li>
<li>Ubbels, G. A., Hara, K., Koster, C. H., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+for+a+functional+role+of+the+cytoskeleton+in+determination+of+the+dorsoventral+axis+in+Xenopus+laevis+eggs%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Evidence for a functional role of the cytoskeleton in determination of the dorsoventral axis in Xenopus laevis eggs.</a> Development. 77 (1), 15-37 (1983).</li>
<li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dejellying+Xenopus+laevis+embryos%5BTitle%5D)+AND+%22CSH+Protoc%22%5BJournal%5D)">Dejellying Xenopus laevis embryos.</a> CSH Protoc. 2007, (2007).</li>
<li>Zahn, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Normal+table+of+Xenopus+development%3A+a+new+graphical+resource%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Normal table of Xenopus development: a new graphical resource.</a> Development. 149 (14), dev200356(2022).</li>
<li>Newport, J., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+major+developmental+transition+in+early+Xenopus+embryos%3A+I.+Characterization+and+timing+of+cellular+changes+at+the+midblastula+stage%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A major developmental transition in early Xenopus embryos: I. Characterization and timing of cellular changes at the midblastula stage.</a> Cell. 30 (3), 675-686 (1982).</li>
<li>Kojima, M. L., Hoppe, C., Giraldez, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+maternal-to-zygotic+transition%3A+reprogramming+of+the+cytoplasm+and+nucleus%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">The maternal-to-zygotic transition: reprogramming of the cytoplasm and nucleus.</a> Nat Rev Genet. 26 (4), 245-267 (2025).</li>
<li>Park, S., Blaser, S., Marchal, M. A., Houston, D. W., Sheets, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+gradient+of+maternal+Bicaudal-C+controls+vertebrate+embryogenesis+via+translational+repression+of+mRNAs+encoding+cell+fate+regulators%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">A gradient of maternal Bicaudal-C controls vertebrate embryogenesis via translational repression of mRNAs encoding cell fate regulators.</a> Development. 143 (5), 864-871 (2016).</li>
<li>Bull, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bicaudal%2C+a+genetic+factor+which+affects+the+polarity+of+the+embryo+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22J+Exp+Zool%22%5BJournal%5D)">Bicaudal, a genetic factor which affects the polarity of the embryo in Drosophila melanogaster.</a> J Exp Zool. 161 (2), 221-241 (1966).</li>
<li>Mohler, J., Wieschaus, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dominant+maternal-effect+mutations+of+Drosophila+melanogaster+causing+the+production+of+double-abdomen+embryos%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Dominant maternal-effect mutations of Drosophila melanogaster causing the production of double-abdomen embryos.</a> Genetics. 112 (4), 803-822 (1986).</li>
<li>Maisonneuve, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bicaudal+C%2C+a+novel+regulator+of+Dvl+signaling+abutting+RNA-processing+bodies%2C+controls+cilia+orientation+and+leftward+flow%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Bicaudal C, a novel regulator of Dvl signaling abutting RNA-processing bodies, controls cilia orientation and leftward flow.</a> Development. 136 (17), 3019-3030 (2009).</li>
<li>Minegishi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluid+flow-induced+left-right+asymmetric+decay+of+Dand5+mRNA+in+the+mouse+embryo+requires+a+Bicc1-Ccr4+RNA+degradation+complex%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires a Bicc1-Ccr4 RNA degradation complex.</a> Nat Commun. 12 (1), 4071(2021).</li>
<li>Maerker, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bicc1+and+Dicer+regulate+left-right+patterning+through+post-transcriptional+control+of+the+Nodal+inhibitor+Dand5%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Bicc1 and Dicer regulate left-right patterning through post-transcriptional control of the Nodal inhibitor Dand5.</a> Nat Commun. 12 (1), 5482(2021).</li>
<li>Piazzon, N., Maisonneuve, C., Guilleret, I., Rotman, S., Constam, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bicc1+links+the+regulation+of+cAMP+signaling+in+polycystic+kidneys+to+microRNA-induced+gene+silencing%5BTitle%5D)+AND+%22J+Mol+Cell+Biol%22%5BJournal%5D)">Bicc1 links the regulation of cAMP signaling in polycystic kidneys to microRNA-induced gene silencing.</a> J Mol Cell Biol. 4 (6), 398-408 (2012).</li>
<li>Kraus, M. R. -C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two+mutations+in+human+BICC1+resulting+in+Wnt+pathway+hyperactivity+associated+with+cystic+renal+dysplasia%5BTitle%5D)+AND+%22Hum+Mutat%22%5BJournal%5D)">Two mutations in human BICC1 resulting in Wnt pathway hyperactivity associated with cystic renal dysplasia.</a> Hum Mutat. 33 (1), 86-90 (2012).</li>
<li>Tran, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+RNA-binding+protein+Bicaudal+C+regulates+polycystin+2+in+the+kidney+by+antagonizing+miR-17+activity%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">The RNA-binding protein Bicaudal C regulates polycystin 2 in the kidney by antagonizing miR-17 activity.</a> Development. 137 (7), 1107-1116 (2010).</li>
<li>Bouvrette, D. J., Sittaramane, V., Heidel, J. R., Chandrasekhar, A., Bryda, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Knockdown+of+Bicaudal+C+in+zebrafish+%28Danio+rerio%29+causes+cystic+kidneys%3A+a+nonmammalian+model+of+polycystic+kidney+disease%5BTitle%5D)+AND+%22Comp+Med%22%5BJournal%5D)">Knockdown of Bicaudal C in zebrafish (Danio rerio) causes cystic kidneys: a nonmammalian model of polycystic kidney disease.</a> Comp Med. 60 (2), 96-106 (2010).</li>
<li>Mahone, M., Saffman, E. E., Lasko, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Localized+Bicaudal-C+RNA+encodes+a+protein+containing+a+KH+domain%2C+the+RNA+binding+motif+of+FMR1%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">Localized Bicaudal-C RNA encodes a protein containing a KH domain, the RNA binding motif of FMR1.</a> EMBO J. 14 (9), 2043-2055 (1995).</li>
<li>Dowdle, M. E., Park, S., Blaser Imboden, S., Fox, C. A., Houston, D. W., Sheets, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+single+KH+domain+in+Bicaudal-C+links+mRNA+binding+and+translational+repression+functions+to+maternal+development%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">A single KH domain in Bicaudal-C links mRNA binding and translational repression functions to maternal development.</a> Development. 146 (10), dev172486(2019).</li>
<li>Saffman, E. E., Styhler, S., Rother, K., Li, W., Richard, S., Lasko, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Premature+translation+of+oskar+in+oocytes+lacking+the+RNA-binding+protein+Bicaudal-C%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Premature translation of oskar in oocytes lacking the RNA-binding protein Bicaudal-C.</a> Mol Cell Biol. 18 (8), 4855-4862 (1998).</li>
<li>Chalabi Hagkarim, N., Grand, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+regulatory+properties+of+the+Ccr4-Not+complex%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">The regulatory properties of the Ccr4-Not complex.</a> Cells. 9 (11), E2379(2020).</li>
<li>Chicoine, J., Benoit, P., Gamberi, C., Paliouras, M., Simonelig, M., Lasko, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bicaudal-C+recruits+CCR4-NOT+deadenylase+to+target+mRNAs+and+regulates+oogenesis%2C+cytoskeletal+organization%2C+and+its+own+expression%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">Bicaudal-C recruits CCR4-NOT deadenylase to target mRNAs and regulates oogenesis, cytoskeletal organization, and its own expression.</a> Dev Cell. 13 (5), 691-704 (2007).</li>
<li>Kugler, J. -M., Chicoine, J., Lasko, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bicaudal-C+associates+with+a+Trailer+Hitch%2FMe31B+complex+and+is+required+for+efficient+Gurken+secretion%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Bicaudal-C associates with a Trailer Hitch/Me31B complex and is required for efficient Gurken secretion.</a> Dev Biol. 328 (1), 160-172 (2009).</li>
<li>Kamenska, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+DDX6-4E-T+interaction+mediates+translational+repression+and+P-body+assembly%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">The DDX6-4E-T interaction mediates translational repression and P-body assembly.</a> Nucleic Acids Res. 44 (13), 6318-6334 (2016).</li>
<li>Ladomery, M., Wade, E., Sommerville, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Xp54%2C+the+Xenopus+homologue+of+human+RNA+helicase+p54%2C+is+an+integral+component+of+stored+mRNP+particles+in+oocytes%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Xp54, the Xenopus homologue of human RNA helicase p54, is an integral component of stored mRNP particles in oocytes.</a> Nucleic Acids Res. 25 (5), 965-973 (1997).</li>
<li>Wühr, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deep+proteomics+of+the+Xenopus+laevis+egg+using+an+mRNA-derived+reference+database%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">Deep proteomics of the Xenopus laevis egg using an mRNA-derived reference database.</a> Curr Biol. 24 (13), 1467-1475 (2014).</li>
<li>Kurien, B. T., Dorri, Y., Dillon, S., Dsouza, A., Scofield, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+overview+of+western+blotting+for+determining+antibody+specificities+for+immunohistochemistry%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">An overview of western blotting for determining antibody specificities for immunohistochemistry.</a> Methods Mol Biol. 717, 55-67 (2011).</li>
<li>Kurien, B. T., Scofield, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Western+blotting%3A+an+introduction%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Western blotting: an introduction.</a> Methods Mol Biol. 1312, 17-30 (2015).</li>
<li>Fisher, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Xenbase%3A+key+features+and+resources+of+the+Xenopus+model+organism+knowledgebase%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Xenbase: key features and resources of the Xenopus model organism knowledgebase.</a> Genetics. 224 (1), iyad018(2023).</li>
<li>Baker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antibody+anarchy%3A+a+call+to+order%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Antibody anarchy: a call to order.</a> Nature. 527 (7579), 545-551 (2015).</li>
<li>Baker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reproducibility+crisis%3A+blame+it+on+the+antibodies%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Reproducibility crisis: blame it on the antibodies.</a> Nature. 521 (7552), 274-276 (2015).</li>
<li>Martin, S. A., Page, S. J., Piccinni, M. Z., Guille, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Confirming+antibody+specificity+in+Xenopus%5BTitle%5D)+AND+%22Cold+Spring+Harb+Protoc%22%5BJournal%5D)">Confirming antibody specificity in Xenopus.</a> Cold Spring Harb Protoc. 2020 (12), (2020).</li>
</ol>

The authors have no conflicts of interest to disclose.

Though simple in concept, immunoblotting is difficult in practice owing to multiple steps spanning several days. As such, achieving consistent, high-quality results is often challenging. Nevertheless, immunoblotting is a vital and ubiquitous component of modern biology. For example, immunoblotting is critical to confirming successful translation and expression of the microinjected mRNA in X. laevis oocytes and embryos, highlighting its importance to X. laevis research. Moreover, immunoprecipitation experiments utilize immunoblotting to analyze in vivo protein-protein interactions. Efficient detection of specific proteins will largely depend upon their abundance and the quality of the antibody detection reagents. In this study, estimated concentrations of the endogenous protein we analyzed range almost 500-fold in the Xenopus egg (Bicc1: 37.22 nM, Cnot1: 115.08 nM, Ddx6: 980.55 nM, Gapdh: 17,907.26 nM)34, but all were detected readily. Below this range, modifications can be made to the workflow at essentially every step of an immunoblot -- from sample collection to imaging -- to improve accuracy and efficiency of detection, if needed.
Which type of SDS-PAGE gel and membrane to use are major considerations for any immunoblotting experiment. In our experience, precast gels are more facile and reproducible than gels produced in-house. Precast gels have the added advantage of being available in gradient formats where the gels gradually increase in polyacrylamide concentration from top to bottom. Gradient gels allow researchers to resolve and analyze a broad range of proteins with vastly different molecular weights. Doing so avoids the typical tradeoff between choosing a low acrylamide percentage gel (&#60;7%), which resolves high molecular weight proteins, versus a high percentage gel (&#62;12%), which is better at resolving low molecular weight proteins. It is also important to consider whether to use a nitrocellulose or polyvinylidene fluoride (PVDF) membrane for protein transfer. The mechanism and advantages of each membrane type have been discussed extensively35,36. In short, PVDF has a higher protein binding capacity (and therefore higher sensitivity) than nitrocellulose membrane and is more durable for repeated usage. However, PVDF requires additional handling in the form of activation by methanol incubation and is more expensive. While we find that both membranes are suitable for most applications, we routinely use nitrocellulose due to cost considerations.
Imaging the immunoblot is a crucial component of the protocol. During this step, researchers must exercise judgment to capture an image of an immunoblot that conveys all the necessary information but keeps background noise to a minimum. There are two main imaging methods for standard HRP-conjugated antibodies: X-ray film or digital imaging systems. In our experience, capturing images with X-ray film has many benefits. X-ray films are generally far more sensitive than digital imaging systems, facilitating the detection of low-abundance proteins. X-ray films also produce a physical record of the experiment that can be annotated and digitized at high resolution. However, a digital imaging system will have a far superior linear range-the relationship between signal strength and image intensity-than a film developer. Due to its sensitivity, an X-ray film can become overexposed quickly, losing quantitative information. Therefore, a researcher looking for precise quantitation of their results should use a digital imaging system or take special care not to overexpose developing immunoblots with X-ray film.
If immunoblotting results are unsatisfactory after following the protocol as we have outlined, such as faint or &#34;blurry&#34; bands upon imaging, multiple adjustments can be made to improve the outcome. For example, the amount of Xenopus protein mixture analyzed can be adjusted. Total protein from as little as one-fifth of an embryo can produce strong results, though it is possible to load up to two embryos or oocytes per gel lane. Loading too much protein may result in artefacts or spurious high background signals during imaging. Additionally, electrophoresis at a lower voltage (100 V) for a longer running time generally leads to sharper protein bands. Similarly, performing the protein transfer overnight at 4&#176;C at low voltage (30 V) instead of an hour at 100 V may improve protein detection, especially for high molecular weight species. Lastly, some primary antibodies incubations detect their targets more robustly using incubations of 1 hour at room temperature, as opposed to 4 &#176;C overnight. This protocol modification must be tested for each primary antibody.
Identifying antibodies to detect endogenous proteins presents a major challenge to immunoblotting in Xenopus, as relatively few Xenopus-specific antibodies are commercially available. Researchers can benefit from non-commercial sources of Xenopus antibodies such as Xenbase37 (https://www.xenbase.org/xenbase/),&#160; the Developmental Studies Hybridoma Bank (https://dshb.biology.uiowa.edu/), and the European&#160;Xenopus Resource Centre (https://xenopusresource.org/). Alternatively, antibodies generated to recognize human or mouse protein may be useful for detecting homologous&#160;Xenopus proteins, depending upon the conservation of the regions used for antibody production. For example, a Bicc1 antibody presented in this protocol was raised against the human Bicc1 protein. For such reagents, the ability to cross-react with Xenopus proteins must be determined empirically. Though the exact epitope companies use to create antibodies are generally proprietary, many will provide an amino acid range for the epitope upon request. This region can then be aligned with the corresponding region of the homologous Xenopus protein. While success is not guaranteed, we have typically had success when the epitope used for antibody production in the target species exhibits at least 75% or above similarity to the comparable stretch of sequence in Xenopus. Antibodies generated against non-Xenopus proteins should be validated before extensive use, as improper verification of antibodies is a major contributor to irreproducible research38,39. This can be done by expressing a tagged Xenopus protein of interest in embryos through mRNA microinjection40. Analyzing the same samples with an antibody towards the affinity tag is then used to confirm that the antibody to the endogenous protein and the antibody to the tagged protein both recognize the same entity (as in Figure 3B).
Analysis of RNA-binding proteins, especially those with regulatory functions such as Bicc1, can prove challenging due to a dearth of reagents and typically low expression of potent regulatory molecules. This problem becomes more apparent in immunoblotting, where each reagent must be optimized separately, and finding antibodies of sufficient specificity and sensitivity may be difficult. Our intent with this protocol is to address specific limitations of immunoblotting in Xenopus and elaborate on steps applicable to other systems, such that researchers may overcome the difficulties of analyzing RNA-binding proteins.

Understanding cellular mechanisms requires monitoring specific protein species. Common inquiries include how they change in expression spatially and temporally, what proteins they interact with, and if they are modified in response to different conditions. Immunoblotting, colloquially known as "Western blotting," is a key methodology to address these challenges. An immunoblot experiment separates proteins from a complex sample-for example, whole cell lysate-and identifies them using antibodies. Specifically, proteins are denatured and then separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) by size. The size fractionated proteins are transferred onto a solid support, such as a nitrocellulose membrane, and the protein of interest is identified using antibody reagents. Immunoblotting has become a standard part of the molecular biologist's toolkit. It is commonly used for monitoring protein expression in different contexts or as an endpoint for experiments such as immunoprecipitation assays. Despite these advantages, analysis of steady-state protein levels with immunoblotting proves challenging, as the assay must be optimized for each step and experimental context (Figure 1).
One challenging context is analyzing material from the African clawed frog Xenopus laevis. X. laevis is an important organism for studying RNA-protein interactions. This system has numerous advantages, chief among them that X. laevis produce hundreds of oocytes and subsequently synchronously developing embryos at a time. Each oocyte has ~25 µg of non-yolk protein1, providing abundant material for biochemical experiments. The large size (~1250 µm)1 of oocytes and embryos also facilitates mRNA microinjection to exogenously express tagged or mutated proteins at scale2. These qualities enable powerful experiments to assay translational control in X. laevis, such as the tethered function assay, in which the impact of a translational regulatory protein on an mRNA can be assayed irrespective of that protein's capacity for RNA binding3,4,5,6. However, immunoblotting in Xenopus oocytes and embryos comes with difficulties, including interference from large masses of yolk platelets in these early cells7, which will obscure results if not removed.
Here, we present an optimized protocol for effective immunoblotting in X. laevis, with an emphasis on detecting translational regulatory proteins. We provide instructions on how to collect and process oocytes and embryos to imaging the final immunoblot. Also included are detailed instructions for optimal protein loading and imaging, as well as preventing yolk contamination of the sample. In addition, we discuss possible protocol modifications and strategies to find antibodies compatible with Xenopus proteins. We intend to provide investigators with guidance to effortlessly perform immunoblotting as part of their own experiments in this underutilized model organism.
<img alt="Xenopus immunoblot flow chart showing sample prep, SDS-PAGE, and antibody incubation process." class="xfigimg" src="/files/ftp_upload/69139/69139fig1.jpg" data-altupdatedat="1783396841000" data-alt="Figure 1"> 
Figure 1: Workflow for Xenopus sample preparation and subsequent immunoblot analysis. <a href="https://www.jove.com/files/ftp_upload/69139/69139fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>100 sq ft Heavy Duty Premium Wrap</td><td>Saran</td><td>2570000130</td><td></td></tr><tr><td>10x Cell Lysis Buffer</td><td>Cell Signaling Technology</td><td>#9803</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148</td><td></td></tr><tr><td>2-Quart Oblong Baking/Serving Dish</td><td>Pyrex</td><td>71160010116</td><td></td></tr><tr><td>2x Laemmli Sample Buffer</td><td>Bio-Rad</td><td>#1610737</td><td></td></tr><tr><td>Anti-HA High Affinity Antibody</td><td>Roche</td><td>11867423001</td><td>Prepared as directed and used at 1:10,000 dilution.</td></tr><tr><td>Anti-Rabbit IgG, HRP-Linked Antibody</td><td>Cell Signaling Technology</td><td>7074S</td><td>Used at 1:30,000 dilution.</td></tr><tr><td>Autoradiography Cassette, 5 x 7 Inches</td><td>Research Products International</td><td>420057</td><td>Not necessary if using a digital imaging system.</td></tr><tr><td>Bel-Art Disposable Pestles</td><td>Millipore Sigma</td><td>BAF199230001</td><td></td></tr><tr><td>Bicaudal-C (Human) Rabbit Antibody</td><td>Envigo Bioproducts (now Inotiv)</td><td>N/A</td><td>Custom ordered. Used at 1:20,000 dilution.</td></tr><tr><td>Bicaudal-C (Xenopus) Rabbit Antibody</td><td>Envigo Bioproducts (now Inotiv)</td><td>N/A</td><td>Custom ordered. Used at 1:20,000 dilution. Ref. Park et al., 2016</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A3294</td><td>A substitute for nonfat milk in blocking buffer.</td></tr><tr><td>Centrifuge 5425</td><td>Eppendorf</td><td>13-864-456</td><td></td></tr><tr><td>CL-XPosure Film</td><td>ThermoFisher Scientific</td><td>34091</td><td>Not necessary if using a digital imaging system.</td></tr><tr><td>CNOT1 (D5M1K) Rabbit Monoclonal Antibody</td><td>Cell Signaling Technology</td><td>#44613</td><td>Used at 1:2,000 dilution.</td></tr><tr><td>DDX6/RCK (D26C11) Rabbit Monoclonal Antibody</td><td>Cell Signaling Technology</td><td>#8988</td><td>Used at 1:5,000 dilution.</td></tr><tr><td>E-Z Store and Pour Developer</td><td>MXR Imaging</td><td>103633</td><td>Not necessary if using a digital imaging system.</td></tr><tr><td>E-Z Store and Pour Fixer</td><td>MXR Imaging</td><td>114511</td><td>Not necessary if using a digital imaging system.</td></tr><tr><td>GAPDH Monoclonal Antibody</td><td>ProteinTech</td><td>60004-1-Ig</td><td>Used at 1:40,000 dilution.</td></tr><tr><td>Gel Releaser</td><td>Bio-Rad</td><td>#1653320</td><td></td></tr><tr><td>Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP</td><td>Invitrogen</td><td>A16066</td><td>Prepared as directed and used at 1:40,000 dilution.</td></tr><tr><td>Ice Pack, 8 oz, 5 x 3 x 1&quot;</td><td>Uline</td><td>S-18256</td><td></td></tr><tr><td>L-Cysteine</td><td>Sigma-Aldrich</td><td>C7352</td><td></td></tr><tr><td>Medical Film Processor</td><td>Konica Minolta</td><td>SRX-101A</td><td>Not necessary if using a digital imaging system.</td></tr><tr><td>Mini-PROTEAN&amp;nbsp;Cassette Opening Lever</td><td>Bio-Rad</td><td>#4560000</td><td></td></tr><tr><td>Mini-PROTEAN&amp;nbsp;Tetra Vertical Electrophoresis Cell</td><td>Bio-Rad</td><td>#1658004</td><td></td></tr><tr><td>MSE PRO 700ml Western Blot Electrophoresis Tank</td><td>MSE Supplies</td><td>BB2369</td><td></td></tr><tr><td>Nitrocellulose Membrane, 0.45 &amp;micro;m</td><td>Bio-Rad</td><td>#1620115</td><td></td></tr><tr><td>Nonfat Dry Milk</td><td>Sanalac</td><td>1570000180</td><td></td></tr><tr><td>Peroxidase AffiniPure Goat Anti-Rat IgG (H+L)</td><td>Jackson Immunoresearch</td><td>112-035-003</td><td>Prepared as directed and used at 1:100,000 dilution.</td></tr><tr><td>Polysorbate (Tween) 20</td><td>Fisher Bioreagents</td><td>BP337-500</td><td></td></tr><tr><td>Ponceau S</td><td>VWR Life Science</td><td>K793-500ML</td><td>An approximation can be made with 0.5% Ponceau S, 1% acetic acid</td></tr><tr><td>PowerPac Basic Power Supply</td><td>Bio-Rad</td><td>1645050</td><td></td></tr><tr><td>PR1MA Variable Speed 2D Rocker</td><td>MidSci</td><td>R2D-30</td><td></td></tr><tr><td>Precision Plus Protein Dual Color Standards Ladder</td><td>Bio-Rad</td><td>#1610374</td><td>Any protein ladder of choice can be used.</td></tr><tr><td>Pure Cellulose Chromatography Filter Paper, 0.35 mm</td><td>Fisher Scientific</td><td>05-714-4</td><td></td></tr><tr><td>Restore Western Blot Stripping Buffer</td><td>ThermoFisher Scientific</td><td>#21059</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Chemical</td><td>S640-10</td><td></td></tr><tr><td>Storage Box 300 mL</td><td>Rosti Mepal</td><td>RST46608</td><td>For nitrocellulose membrane blocking, antibody incubation, and washes</td></tr><tr><td>SuperSignal West Femto Maximum Sensitivity Substrate</td><td>ThermoFisher Scientific</td><td>#34094</td><td>An enhanced chemiluminescent (ECL) substrate containing two components: luminol and enhancer. This ECL reagent has a low-femtogram sensitivity.</td></tr><tr><td>SuperSignal West Pico PLUS Chemiluminescent Substrate</td><td>ThermoFisher Scientific</td><td>#34580</td><td>An enhanced chemiluminescent (ECL) substrate containing two components: luminol and enhancer. This ECL reagent has a picogram to femtogram sensitivity.</td></tr><tr><td>SurePAGE, Bis-Tris, 10 x 8, 4&amp;ndash;12%, 10 Well Polyacrylamide Gels</td><td>GenScript</td><td>M00652</td><td></td></tr><tr><td>Transfer Buffer Powder</td><td>GenScript</td><td>M00652</td><td></td></tr><tr><td>Tris Base Ultrapure</td><td>USBiological</td><td>77-86-1</td><td></td></tr><tr><td>Tris-MOPS-SDS Running Buffer Powder</td><td>GenScript</td><td>M00138</td><td></td></tr></tbody></table>

optimized analysis of proteins from xenopus oocytes and embryos by immunoblotting

Optimized Analysis of Proteins from Xenopus Oocytes and Embryos by Immunoblotting

Analysis of proteins by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting (western blotting) is a vital part of the molecular biologist's toolkit. This technique separates a complex protein mixture by molecular weight and then assays the presence of target proteins using specific antibodies. Immunoblotting has a variety of applications. Examples include use as a targeted approach to study protein-protein interactors or as a control to confirm expression or depletion of protein targets. However, the successful execution of immunoblotting requires complicated, multistep experiments. Protocols must be optimized for each organism, target protein, and application. Therefore, knowledge gaps exist for the use of immunoblots in many models, including the model frog Xenopus laevis.
Due to their large size, abundant material for biochemical experiments, and facile handling, X. laevis oocytes and embryos have been vital for studying principles of translational control. However, this species lacks specific protocols for robust and routine immunoblotting. Here, we offer an in-depth protocol for western blotting optimized for samples from multiple Xenopus developmental stages. We then analyze translational regulators across development.

To demonstrate the efficiency of our immunoblotting protocol, we analyzed affinity-tagged and endogenous translational control proteins in three types of X. laevis samples: stage VI oocytes, stage 7 embryos (blastula), and stage 10.5 embryos (gastrula). These stages were chosen because they span the mid-blastula transition (stage 8.5), which marks the beginning of robust zygotic transcription13,14. Because development prior to the mid-blastula transition occurs in the absence of transcription, pre-zygotic (i.e., maternally-controlled) embryos rely heavily on translational control mechanisms to express cell fate proteins at the correct temporal and spatial resolution15. Therefore, the mid-blastula transition is a vital context in which to study RNA-protein interactions.
To analyze protein expression via immunoblotting, X. laevis oocytes and embryos were injected with 500 pg of mRNA encoding the C-terminal half of X. laevis Bicaudal-C (Bicc1) fused to 3x hemagglutinin (HA) affinity tags. We chose to analyze Bicc1 due to its relevance to Xenopus biology and protein-RNA interactions. Bicc1 is an mRNA-binding protein and translational repressor that guides cell fate decisions in the early embryo16,17,18. Later in development, it impacts left-right patterning19,20,21 and the function of organs, including the kidneys22,23,24,25. Bicc1 contains a region that directs translational repression5 and hnRNP K homology (KH) domains that mediate binding to specific mRNAs5,26,27,28.
Extracts were prepared from five HA-Bicc1-injected oocytes or embryos, along with corresponding uninjected samples as negative controls. One tenth of each extract was electrophoresed on a 4-12% gradient bis-tris gel and wet transferred onto a nitrocellulose membrane. Ponceau staining of the membrane to detect total protein confirmed successful transfer (Figure 3A). As part of our studies, we generated an antibody in rabbits that recognizes the C-terminal half of the human Bicc1 protein. This antibody was used to detect endogenous and exogenous Xl-Bicc1 protein (Figure 3B). Previous work showed that Bicc1 protein is absent from X. laevis oocytes, but is expressed in pre-MBT embryos, before the zygotic genome is active16. This suggests that while Bicc1 mRNA accumulates during oogenesis, it is translationally repressed. In agreement with this previous work, endogenous Bicc1 was not detected in oocytes. In contrast, the antibody recognized endogenous Bicc1 (~MW 107 kDa) in both stage 7 and 10.5 embryos. Taken together, these results validate that the antibody recognizes the endogenous Bicc1 protein (Figure 3B, top panel, red arrowhead). The Bicc1 antibody recognized a smaller protein of approximately 70 kDa in extracts prepared from mRNA-injected samples (Figure 3B, top panel, black arrowhead, compare lanes 2, 4, and 6 with lanes 1, 3, and 5). As a control for the specificity of the Bicc1 antibody, the membrane was stripped and re-probed with an antibody against the HA affinity tag. The HA antibody identified the 70 kDa protein in the injected samples but did not recognize the endogenous Bicc1 (Figure 3B, second panel, black arrowhead). Detection of the HA-Bicc1 C-term varies in intensity across stages due to differences in protein expression from injected mRNA in the different cell types.
Next, we expanded the results by testing the expression of endogenous translational regulatory proteins that interact with Bicc1. First, we analyzed the expression of Ccr4-Not Transcription Complex Subunit 1 (Cnot1), a large scaffold protein and part of the Ccr4-Not deadenylase (CNOT) complex. The multi-subunit CNOT complex is one of the principal eukaryotic deadenylases and is primarily known for trimming the poly(A) tail at the end of messenger mRNAs as a prelude to their degradation. However, this complex has diverse roles in translational control that extend beyond deadenylation29. We were interested in assaying Cnot1 expression due to the CNOT complex's importance to mRNA regulation and previous observations that Cnot1 interacts with Bicc120,30. To analyze Cnot1 expression, we used an antibody that recognizes the endogenous Cnot1 protein. It identified two proteins at 250 kDa and 270 kDa in each sample, the predicted size of Cnot1 (Figure 3B, third panel). Thus, this protocol allows us to readily identify a critical component of a regulatory complex. In addition, this data supports the protocol's ability to successfully identify high molecular weight proteins.
To further test the generalizability of these results, we next analyzed samples for the presence of a prominent DEAD-box helicase involved in translational repression, DEAD-box helicase 6 (Ddx6, previously known as xp54 in Xenopus and me31b in Drosophila). Ddx6 is an important component of ribonucleoprotein granules, is involved in mRNA storage, and interacts with Bicc1 and Cnot16,31,32. An antibody recognizing the endogenous Ddx6 identified a protein of around 54 kDa in each sample (Figure 3B, fourth panel). Expression was reduced in zygotic embryos (Figure 3B, compare lanes 5 and 6 to 1-4), as reported previously33.
Finally, to ensure that the differences we observed between samples were due to differences in expression, we stripped and re-probed the immunoblot with an antibody recognizing Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) enzyme. Gapdh serves as a ubiquitously expressed "loading control." Equivalent levels of Gapdh expression confirm the Ponceau staining: an equal amount of total protein is being analyzed across samples (Figure 3B, fifth panel).
Together, these results confirm the efficacy of this immunoblot method. We were able to identify exogenous and endogenous Bicc1 across multiple X. laevis developmental stages relevant to the study of RNA-protein interactions: oocytes and pre-MBT embryos, which contain only maternally deposited mRNAs, and post-MBT embryos, which also include zygotically transcribed mRNAs. We were able to generalize these results by analyzing the expression of multiple translational control proteins at a variety of molecular weights (Bicc1, Cnot1, and Ddx6) and a loading control (Gapdh). We also show that this protocol can be used for X. tropicalis embryos with modification to increase the amount of material used to account for their smaller size (Supplementary Figure 1).
<img alt="Ponceau staining and immunoblot analysis; protein detection; experimental results; stage comparison." class="xfigimg" src="/files/ftp_upload/69139/69139fig3.jpg" data-altupdatedat="1783396841000" data-alt="Figure 3"> 
Figure 3: Identifying levels of translational regulatory proteins through immunoblotting in X. laevis. (A) Ponceau staining of the immunoblot to identify total protein from X. laevis stage VI oocytes, stage 7 embryos, and stage 10.5 embryos. Each lane represents 10% of the protein prepared from an extract (0.5 oocyte or embryo). (B) Immunoblot analysis of selected X. laevis translational regulatory proteins in stage VI oocytes, stage 7 embryos, and stage 10.5 embryos. Lanes 2, 4, and 6 correspond to samples microinjected with HA-Bicc1 mRNA prior to analysis, while lanes 1, 3, and 5 serve as negative (uninjected) controls. α-Bicc1 antibody was used to assay expression of endogenous Bicc1 across stages (top panel). The blot was subsequently stripped and re-probed with an antibody to the HA affinity tag as a control for α-Bicc1 antibody specificity (second panel). Blots were stripped and re-probed for additional regulatory proteins using α-Cnot1 (third panel) and α-Ddx6 (fourth panel). α-Gapdh (bottom panel) served as a control for equal protein loading. Red arrowheads mark the location of endogenous Bicc1, while black arrowheads mark the location of exogenously expressed HA-Bicc1 C-term. <a href="https://www.jove.com/files/ftp_upload/69139/69139fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Immunoblot analysis of Bicc1 protein in Xenopus laevis versus Xenopus tropicalis. α-Bicc1 antibody was used to assay expression of endogenous and exogenous Bicc1 across different amounts of X. laevis and X. tropicalis embryo extract. Lane 1: 0.5 uninjected X. laevis embryo. Lane 2: 0.5 HA-Bicc1 injected X. laevis embryo. Lane 3: 3 uninjected X. tropicalis embryos. Lane 4: 1 uninjected X. tropicalis embryo. Lane 5: 0.5 uninjected X. tropicalis embryo. Two antibodies recognizing endogenous Bicc1 were used: one raised against human Bicc1 (top panel) and the other raised against X. laevis Bicc1 (middle panel). α-Gapdh (bottom panel) was used to control for equal protein loading. <a href="https://www.jove.com/files/ftp_upload/69139/Supplementary%20Figure%201.pdf" target="_blank">Please click here to download this File.</a>

This article describes a protocol for analyzing proteins from Xenopus oocytes and embryos by immunoblotting. Collection steps are described, followed by steps corresponding to sample processing, SDS-PAGE, transfer, antibody staining, and imaging. The protocol emphasizes studying translational regulatory protein complexes with endogenous antibodies and antibodies against protein affinity tags.

Analysis of proteins by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting (western blotting) is a vital ...

Over the course of an animal's lifetime, sulfate decisions occur continually, that allow for normal development and the health an adult organism. These decisions depend on specific proteins referred to sulfate regulators. The goal of our research is to find the mechanisms that control the synthesis of these critical regulators.In addition to providing a detailed outline of immunoblotting and Xenopus oocytes and embryos, our protocol gives insight into challenges common to this model organism, such as antibody procurement. Through in-depth study of the binding and repression mechanisms of Bicaudal C, our research has helped establish a foundation of information by which to compare other translational regulators. To begin processing cells for immunoblotting, add 10 microliters of chilled cell lysis buffer diluted to one-times concentration with double deionized water per embryo or oocyte.Using a micropestle, homogenize the samples on ice. Place the tubes in a benchtop centrifuge and spin the samples at 5, 000 g at four degrees Celsius for 10 minutes to pellet debris, including yolk and insoluble pigments. Transfer the supernatant to a clean 1.5-milliliter tube, ensuring that the pellet is not disturbed during transfer.Add an equal volume of two-times Laemmli sample buffer supplemented with 5%weight by volume 2-mercaptoethanol to the collected supernatant. Heat the mixture at 95 to 100 degrees Celsius for 10 minutes to denature the proteins. Now, remove a 4 to 12%bis-tris SDS precast gel from its packaging and peel off the sticker from the bottom of the gel.Insert the gel into the electrode assembly opposite a buffer dam, and place the entire assembly into a vertical electrophoresis tank. Then, fill both the electrode assembly and the bottom of the tank with Tris-MOPS-SDS running buffer. Gently remove the gel comb and pipette running buffer into the wells to eliminate bubbles and equilibrate the buffer.Now, load five microliters of pre-stained protein standards into the first well and 10 microliters of each prepared sample into the remaining wells. Place the lid on the electrophoresis tank and connect it to a power supply. Then, set the voltage to 200 volts to initiate electrophoresis.Stop electrophoresis once the sample-buffer dye front exits the gel. Before electrophoresis completes, begin assembling the transfer sandwich in the transfer clip. Fill a glass baking dish with chilled transfer buffer and submerge all transfer materials in this buffer during assembly.Place the transfer clip in the dish with the black half lying against the bottom, the white half pointed upward, and the clip opened to the left. Lay one or two fiber sponges flat on the black half of the transfer clip. After cutting two pieces of 0.35-millimeter cellulose chromatography paper to size, place one on top of the sponges.Once electrophoresis is complete, remove the gel from the electrophoresis tank. Using the gel cassette opening lever, carefully pry apart the gel plates, while ensuring that the gel remains attached to one side. Trim off the wells from the top of the gel using the gel releaser.Place the gel, still attached to one half of the plastic cassette, onto the filter paper, and remove the remaining cassette half so the gel stays flat on the paper. Next, cut a piece of 0.45-micrometer nitrocellulose membrane to match the gel's dimensions. Remove the membrane from its protective paper, briefly wet it in transfer buffer, and carefully place it on top of the gel.Then, lay a second piece of filter paper on top of the nitrocellulose membrane. Using a roller, gently but firmly press out all air bubbles. Stack one or two additional fiber sponges on top of the filter paper to complete the sandwich.Close the transfer cassette and clip it tightly to seal the assembled sandwich. Then, insert the closed transfer sandwich into the transfer core with the clip facing upwards, and ensure the black side of the clip faces the black side of the core. Place the transfer core into the electrophoresis tank.Add an ice pack and a stir bar to the tank, ensuring the stir bar can rotate freely. Fill the tank with chilled transfer buffer until the apparatus is fully submerged, and secure the lid on top. Connect the apparatus to the power supply, match electrode colors, and set the conditions to 100 volts for one hour with the stir bar rotating.Once the transfer is complete, carefully open the cassette and disassemble the sandwich. Remove the nitrocellulose membrane and mark the side that was in contact with the gel. Then, place the nitrocellulose membrane in a small container so that it lies flat against the bottom.Cover the membrane completely with Ponceau stain and rock gently on a rocker for 5 to 10 minutes. After pouring the Ponceau stain, rinse the membrane several times with double deionized water until the excess stain is removed and protein bands become visible in the lanes. Then, perform membrane blocking, antibody incubation, and washing steps as shown.Once the secondary antibody incubation and washing are complete, the membrane is ready for imaging. In a dark room, turn on the film developer and allow it to warm up. Cut two squares of plastic wrap large enough to fully cover the membrane.Using forceps, place the nitrocellulose membrane face up onto the first square of plastic wrap. In a 1.5-milliliter tube, mix equal volumes of the enhanced chemiluminescence luminol and enhancer reagents. Then, use approximately one milliliter of the mixture to cover most membranes.Using the plastic wrap, gently manipulate the membrane to ensure the entire surface is evenly coated and incubate for one minute. Then, remove excess ECL reagent by grasping the membrane with forceps and lightly shaking off the liquid. Place the membrane face up onto the second square of plastic wrap, fold it loosely over the membrane, and tape the wrap securely into an autoradiography cassette.Next, image the membrane using an autoradiography film in a dark room by following the steps shown. After desired protein visualization is achieved, align the developed film with the glow-in-the-dark markers, and use them as a guide to mark the position of the pre-stained protein standards onto the film. Once imaging is complete, carefully remove the membrane from the plastic wrap and immerse it in TBSTW to wash off residual ECL reagent.Ponceau staining of the membrane confirmed successful protein transfer. Endogenous B1 protein was not detected in oocyte samples, but was clearly detected in stage seven and stage 10.5 embryo samples at approximately 107 kilodaltons. A smaller 70-kilodalton protein band was detected in injected samples across all stages using the Bicc1 antibody, indicating successful expression of the HA-Bicc1 C-terminal fusion.The HA antibody detected the same 70-kilodalton band only in injected samples, confirming the specificity of the Bicc1 antibody for the fusion protein. Two high-molecular-weight bands, approximately 250 and 270 kilodaltons, were detected in all samples using the CNOT1 antibody. Expression of the 54-kilodalton DDX6 protein was detected in all samples, but was visibly reduced in stage 10.5 embryos.

optimized-analysis-proteins-from-xenopus-oocytes-embryos

Research

JoVE Journal

Biochemistry

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition.  This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Immunoblot Analysis

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

The question of how chromatin regulators and chromatin states affect the genome in vivo is key to our understanding of how early cell fate decisions are made in the developing embryo. ChIP-Seq—the most popular approach to investigate chromatin features at a global level—is outlined here for Xenopus embryos.

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus laevis as a Model to Identify Translation Impairment

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Zebrafish targeting reactive electrophiles and oxidants (Z-REX) is a chemical biology-based method for the investigation of reactive small-molecule signaling. This technique can be applied to live fish of different developmental stages. Here, we couple standard assays in zebrafish with Z-REX for signaling pathway analysis.

Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites

Learn immunoblotting techniques for protein analysis in Xenopus oocytes and embryos. This protocol enables detection of translational regulators and sulfate regulatory proteins.

Optimized Analysis of Proteins from Xenopus Oocytes and Embryos by Immunoblotting

Explore More Videos