Name: In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions (Video) | JoVE Journal
Uploaded: 2019-07-17T14:00:00.000+00:00
Duration: 8 min 14 s
Description: Learn biotin-labeled biochemical assays for studying protein-nucleic acid interactions. This robust, efficient method enables reproducible experiments without special equipment and can be completed within a single day.

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5 minutes

We thank P. Jeremy Wang (University of Pennsylvania) for helpful edits and discussions. We also thank Sigrid Eckardt for language editing. K. Z. was supported by National Key R&#38;D Program of China (2016YFA0500902, 2018YFC1003500) and National Natural Science Foundation of China (31771653). L. Y. was supported by National Natural Science Foundation of China (81471502, 31871503) and Innovative and Entrepreneurial Program of Jiangsu Province.&#160;J. N. was supported by Zhejiang Medical Science and Technology Project (2019KY499).&#160;M. L. was supported by grants of National Natural Science Foundation of China (31771588) and the 1000 Youth Talent Plan.

1. Protein preparation
<ol>
	<li>MEIOB and MOV10 expression constructs
	<ol>
		<li>Generate cDNA expression constructs encoding mouse MEIOB-A, C, and E (Figure 1A) and MOV10.
		<ol>
			<li>Set up the polymerase chain reaction (PCR) reactions for each fragment. Mix 1 &#956;L of mouse cDNA (from C57BL/6 mouse testis), 1 &#956;L of dNTP, 2 &#956;L of 10 &#956;M forward primer, 2 &#956;L of 10 &#956;M reverse primer, 1 &#956;L of DNA polymerase, 25 &#956;L of 2x PCR buffer, and 18 &#956;L of double distilled H2O (ddH2O) in a final volume of 50 &#956;L. 
			NOTE: The primers for the amplification of Meiob and Mov10 gene fragments are listed in Table 1.</li>
			<li>Perform PCR reactions using the following programs: 95 &#176;C for 5 min, 35 cycles of heating at 95 &#176;C for 15 s, annealing at 64 &#176;C for 15 s, extension at 72 &#176;C for 20 s (2 min for extending full length MOV10), and final extension at 68 &#176;C for 7 min. 
			NOTE: Use primer pair MEIOB-E (F1) and MEIOB-E-mut (R1) and MEIOB-E-mut (F2) and MEIOB-E (R2) to amplify two segments that contain the mutant sequence within an overlapping sequence at the 3&#39;&#160;and 5&#39;&#160;ends, respectively, to generate a mutant PCR template.</li>
		</ol>
		</li>
		<li>Analyze the amplified PCR DNA by gel electrophoresis, cut the band of required size from the gel quickly under a UV lamp, and place into a centrifuge tube. 
		NOTE: The expected product sizes visible on the agarose gel for MEIOB-A is 536 bp, MEIOB-C is 296 bp, MEIOB-E are 312 bp and 229 bp, and MOV10 is 3015 bp.</li>
		<li>Purify the PCR DNA with a gel extraction kit following the manufacturer&#39;s protocol.
		<ol>
			<li>Add an equal volume of dissolving buffer into the centrifuge tube from step 1.1.2 and melt gel in a 50-55 &#176;C water bath for 5-10 min, ensuring that the gel pieces melt completely. Centrifuge briefly to collect any droplets from the wall of the tube. 
			NOTE: The mass/volume concentration of the gel and the dissolving buffer is 1 mg/&#956;L.</li>
			<li>Place the adsorption column in the collection tube, transfer the solution containing the dissolved gel fragment to the adsorption column, and centrifuge at 12,000 x g for 2 min.</li>
			<li>Discard the filtrate at the bottom of the collection tubes. Add 600 &#956;L of the wash buffer to the column, centrifuge at 12,000 x g for 1 min, and discard the filtrate.</li>
			<li>Repeat step 1.1.3.3 once.</li>
			<li>Place the column back into the collection tube, and centrifuge at 12,000 x g for 2 min to remove all the remaining wash buffer.</li>
			<li>Place the adsorption column in a 1.5 mL sterilized centrifuge tube, add 50 &#956;L of ddH2O to the center of the adsorption column and centrifuge at 12,000 x g for 1 min. Measure the DNA concentration of the eluate using spectrophotometer.</li>
		</ol>
		</li>
		<li>Restriction digestion of plasmids
		<ol>
			<li>Digest pGEX-4T-1 vector with BamHI and NotI. To do so, mix 4 &#956;g of pGEX-4T-1 vector, 5 &#956;L of 10x digest buffer, 1 &#956;L of BamHI, and 1 &#956;L of NotI and ddH2O to a final reaction volume of 50 &#956;L. Incubate at 37 &#176;C for 2 h.</li>
			<li>Digest pRK5 vector with BamHI and XhoI by mixing 4 &#956;g of pRK5 vector, 5 &#956;L of 10x digest buffer, 1 &#956;L of BamHI, and 1 &#956;L of XhoI and ddH2O to a final reaction volume of 50 &#956;L. Incubate at 37 &#176;C for 2 h.</li>
		</ol>
		</li>
		<li>Analyze the vector DNA by gel electrophoresis, cut the desired size band from the gel quickly with a scalpel under a UV lamp and place it into a centrifuge tube.</li>
		<li>Purify the vector DNA with a gel extraction kit as 1.1.3 following the manufacturer&#39;s instruction. 
		NOTE: The length of linearized plasmids: pGEX-4T-1, 4.4 kb; pRK5, 4.7 kb.</li>
		<li>Set up a standard recombinant ligation reaction by combining 0.03 pmol of linearized vector, 0.06 pmol of cDNA fragment, 2 &#956;L of ligase, and 4 &#956;L of 5x ligase buffer and ddH2O in a final reaction volume of 10 &#956;L. 
		NOTE: Clone MEIOB-A, C, and E into a pGEX-4T-1 vector and MOV10 into a pRK5 vector.</li>
		<li>Incubate the mixture at 37 &#176;C for 30 min, and then cool the reaction immediately for 5 min on ice. Transform MEIOB recombinant plasmids into BL21 bacteria and MOV10 recombinant plasmids into DH5&#945;&#160;bacteria. 
		NOTE: Verify all recombinant constructs by Sanger sequencing.</li>
		<li>Prepare glycerol stocks of bacterial cultures containing verified recombinant plasmids by adding an equal volume of 50% glycerol to liquid cultures, and store at -80 &#176;C. 
		NOTE: For each subsequent experiment, streak out bacteria from glycerol stocks onto a fresh agar plate and use a single colony for the expansion as described in step 1.2.</li>
	</ol>
	</li>
	<li>MEIOB protein extracts from bacteria
	<ol>
		<li>Pick one colony of each BL21 strain transfected with the empty or recombinant pGEX-4T-1 plasmid verified by sequencing and inoculate in 3 mL LB containing 100 &#181;g/mL ampicillin. Grow overnight at 37 &#176;C with shaking at 220 rpm.</li>
		<li>Inoculate 300 mL LB containing 100 &#181;g/mL ampicillin from 3 mL overnight culture (from step 1.2.1). Grow the cultures with shaking at 37 &#176;C for 2 h till OD600 reaches 0.5-1.0.</li>
		<li>Induce the protein expression by adding isopropyl beta-D-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM. Incubate the cultures for an additional 3 h with shaking at 180 rpm and 18 &#176;C.</li>
		<li>Centrifuge the bacterial culture at 3,500 x g and 4 &#176;C for 20 min.</li>
		<li>Resuspend the pellet in 20 mL of ice-cold Dulbecco&#39;s phosphate buffered saline (DPBS) buffer. Sonicate the bacterial suspension on ice for 25 cycles in short 10 s bursts (output power 20%) followed by 2-3 s resting on ice.</li>
		<li>Centrifuge the lysate at 12,000 x g and 4 &#176;C for 15 min. Transfer all the supernatant to a fresh tube.</li>
		<li>Pre-wash beads.
		<ol>
			<li>Add 300 &#956;L of glutathione-sepharose beads to a fresh 15&#160;mL tube and wash the beads with 10 mL of ice-cold PBS buffer.</li>
			<li>Centrifuge at 750 x g and 4 &#176;C for 1 min to pellet the beads and discard the wash solution.</li>
		</ol>
		</li>
		<li>Add the lysate to the washed beads and incubate at 4 &#176;C for 2 h. Centrifuge at 750 x g and 4 &#176;C for 1 min to pellet the beads. Rinse the beads in 10 mL of ice-cold PBS 8x.</li>
		<li>Elute the beads with 1 mL of the elution buffer (10 mM glutathione in 50 mM Tris-HCl at pH 8.0) 6x, incubating at 4 &#176;C for 10 min prior to each elution step.&#160;Centrifuge at 750 x g and 4 &#176;C for 1 min to pellet the beads.&#160;Collect and pool the 6 fractions.</li>
		<li>Transfer the eluted proteins into a centrifugal filter and concentrate by centrifugation at 7,500 x g to obtain a final volume of 100-200 &#956;L.</li>
	</ol>
	</li>
	<li>MOV10 protein extracts from HEK293T cells
	<ol>
		<li>Transiently express the MOV10 proteins in cultured HEK293T cells.
		<ol>
			<li>Prepare MOV10-pRK5 plasmid at a concentration &#62;500 ng/&#956;L.</li>
			<li>Seed HEK293T cells in 15 cm dishes. When the cell density reaches ~70%-90%, replace the cell culture medium with fresh Dulbecco&#39;s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.</li>
			<li>For one transfection, dilute 60 &#956;g of MOV10-pRK5 plasmid DNA in 3 mL of the reduced serum medium, then add 120 &#956;L of the transfection enhancer reagent, and mix well.</li>
			<li>In a separate tube dilute 90 &#956;L of the transfection reagent with 3 mL of reduced serum medium (without penicillin-streptomycin) and mix well.</li>
			<li>Add the diluted DNA to each tube of diluted transfection reagent. Incubate at room temperature for 15 min.</li>
			<li>Add the transfection mixture to the cell culture, and culture cells for ~36-48 h.</li>
		</ol>
		</li>
		<li>After 36-48 h, collect cells from each plate in a 50 mL tube. Centrifuge at 500 x g for 5 min at 4 &#176;C. Wash each pellet with 10 mL of ice-cold PBS, and collect cells by centrifugation at 500 x g for 5 min at 4 &#176;C.</li>
		<li>Resuspend the pellet in 3 mL of cell lysis buffer containing complete ehylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail. Incubate for 30 min on ice. Centrifuge the lysate at 20,000 x g and 4 &#176;C for 20 min.</li>
		<li>Add 100 &#956;L of anti-FLAG magnetic beads per dish of cells in a 1.5 mL tube.</li>
		<li>Wash the magnetic beads 2x with K150 buffer (50 mM HEPES at pH 7.5, 150 mM KoAc, 1 mM DTT, 0.1% NP-40).
		<ol>
			<li>Resuspend the magnetic beads in 1 mL of ice-cold K150 buffer.</li>
			<li>Incubate the magnetic beads for 2 min at 4 &#176;C with gentle rotation and pellet the magnetic beads with the help of a magnet. Remove and discard the supernatant.</li>
		</ol>
		</li>
		<li>Add the magnetic beads to the cell lysate supernatant from step 1.3.3 and incubate at 4 &#176;C for 2 h.</li>
		<li>Wash the protein bound magnetic beads 3x with K150 buffer, then 2x with K150 containing 250 mM NaCl, then 3x with K150 buffer as 1.3.5.</li>
		<li>Resuspend the beads in 300 &#956;L of FLAG elution buffer (100 mM NaCl, 20 mM Tris-HCl at pH 7.5, 5 mM MgCl2, 10% glycerol), add FLAG peptide to a final concentration of 0.5 &#956;g/&#956;L, and incubate with beads on a rotator at 4 &#176;C for 1 h, then pellet the magnetic beads with the help of a magnet.</li>
		<li>Collect the supernatant which contains the eluted MOV10 proteins, determine the concentration, and store at -80 &#176;C for future use.</li>
	</ol>
	</li>
</ol>
2. Nucleic acid preparation
<ol>
	<li>Purchase DNA and RNA oligonucleotides (oligos) with or without biotin labels from a suitable source. Dilute each oligo in RNase-free ddH2O to 20 &#956;M and keep it at -80 &#176;C for the future use. 
	NOTE: The oligo sequences of DNA/RNA substrates used in this study are listed in Table 2.</li>
	<li>Prepare the following mixture for the double-stranded RNA (dsRNA) annealing reaction for MOV10 helicase activity assay: mix 60 mM N-2-hydroxyethylpiperazine-N-ethane-sulphonic acid (HEPES) at pH 7.5, 6 mM KCl, 0.2 mM MgCl2 and RNase-free ddH2O in a final reaction volume of 20 &#956;L.</li>
	<li>Anneal RNA oligos to form RNA duplex by heating a mixture of the biotin-labeled top strand (2 &#956;M, final concentration) and a 1.5-fold of its unlabeled complementary bottom strand in the annealing buffer (step 2.2) at 95 &#176;C for 5 min, and then slowly cool it to room temperature (RT).</li>
</ol>
3. In vitro biochemical assays
<ol>
	<li>EMSA and enzymatic reactions
	<ol>
		<li>For the MEIOB EMSA assay, mix 50 mM Tris HCl at pH 7.5, 2 mM MgCl2, 50 mM NaCl, 10 mM EDTA, 2 mM dithiothreitol (DTT), 0.01% NP-40, 0.8 mM (or other relevant concentrations as shown in Figure 2 and Figure 3) MEIOB protein, and 10 nM biotin-labeled oligonucleotides and ddH2O in a final reaction volume of 20 &#956;L. Incubate at RT for 30 min, and add 5x stop buffer (125 mM EDTA, 50% glycerol) to stop the reaction. &#160;</li>
		<li>Set up MEIOB nuclease activity reactions as described in step 3.1.1 but without the addition of 10 mM EDTA.</li>
		<li>For the MOV10 helicase activity assay, mix 50 mM Tris-HCl at pH 7.5, 20 mM KoAc, 2 mM MgCl2, 0.01% NP-40, 1 mM DTT, 2 U/&#956;L RNase inhibitor, 10 nM biotin-labeled RNA substrate, 2 mM adenosine triphosphate (ATP), 100 nM RNA trap, and 20 ng of MOV10 protein and ddH2O in a final reaction volume of 20 &#956;L. Incubate the reaction mixture at 37 &#176;C for 10 min, 30 min, and 60 min. Add the 5x stop buffer to stop the reaction. 
		NOTE: RNA trap, a biotin-unlabeled oligo with sequence, complementarity to the labeled oligo, which prevents the unwound dsRNA from annealing again.</li>
	</ol>
	</li>
	<li>Polyacrylamide gels
	<ol>
		<li>Wash the gel plates (16 cm x 16 cm) and 1.5 mm combs. Assemble the gel electrophoresis units.</li>
		<li>To prepare a 10% native polyacrylamide gel, mix 14 mL of ddH2O, 1.25 mL of 10x Tris-boric acid-EDTA (TBE), 8.3 mL of 30% acrylamide, 1.25 mL of 50% glycerol, 187.5 &#956;L of 10% freshly prepared ammonium persulfate (APS), and 12.5&#160;&#956;L of tetramethylethylenediamine (TEMED).</li>
		<li>To prepare a 20% native polyacrylamide gel, mix 5.5 mL of ddH2O, 1.25 mL of 10x TBE, 1.25 mL of 50% glycerol, 16.7 mL of 30% acrylamide, 187.5 &#956;L of 10% APS, and 12.5 &#956;L of TEMED.</li>
		<li>Pour the acrylamide solution immediately to the gel and insert the comb. Let the mixture polymerize for approximately 30 min.</li>
	</ol>
	</li>
	<li>Gel running
	<ol>
		<li>Remove the comb, fill the tanks with the electrophoresis running buffer (0.5x TBE).</li>
		<li>Rinse the sample wells with 0.5x TBE buffer, then pre-run the gel at 100 V on ice for 30 min. Replace the running buffer with fresh 0.5x TBE.</li>
		<li>Load 20-25 &#956;L samples into each well.</li>
		<li>Use a 10% native acrylamide gel for the EMSA assay and a 20% native acrylamide gel for the enzymatic assays. Run electrophoresis at 100 V on the ice bath until the bromophenol blue marker has migrated to the bottom quarter of the gel.&#160;</li>
	</ol>
	</li>
	<li>Disassemble the gel plates, trim the gel by removing loading wells and unused lanes. Place the gel in 0.5x TBE buffer.</li>
	<li>Cut the filter paper and the nylon membrane to the size of the gel. Pre-wet the clean filter paper and the nylon membrane.</li>
	<li>Assemble the stack for transfer.
	<ol>
		<li>Place the pre-wet membrane onto the pre-wet filter paper.</li>
		<li>Place the gel on the membrane.</li>
		<li>Cover the gel with another layer of pre-wet filter paper.</li>
		<li>Remove all air bubbles by rolling a clean pipette from center to edge.</li>
	</ol>
	</li>
	<li>Transfer the samples from the gel to the membrane in a semi-dry electrophoretic apparatus at 90 mA for 20 min.</li>
	<li>Stop the transfer, and then dry the membrane on a new filter paper for 1 min.</li>
	<li>Crosslink the samples by irradiating the membrane at 120 mJ/cm2 for 45-60 s in a UV-light crosslinker equipped with 254 nm bulbs (auto crosslink function). Air dry the membrane at RT for 30 min.</li>
	<li>Chemiluminescence detection
	<ol>
		<li>Protocol 1: Use a standard volume of commercial chemiluminescent nucleic acid detection kit.
		<ol>
			<li>Add 20 mL of blocking buffer to the membrane and incubate for 15-30 min with gentle shaking on a rotator at 20-25 rpm.</li>
			<li>Prepare conjugate/blocking buffer solution by adding 66.7 &#956;L stabilized streptavidin-horseradish peroxidase conjugate to 20 mL of blocking buffer.</li>
			<li>Gently remove the blocking buffer and replace it with conjugate/blocking buffer. Incubate for 15 min on a rotator at 20-25 rpm.</li>
			<li>Wash the membrane 4x with shaking at 40-45 rpm for 5 min each.</li>
			<li>Add 30 mL of substrate equilibration buffer to the membrane. Incubate the membrane for 5 min with shaking at 20-25 rpm.</li>
			<li>Prepare substrate working solution by adding 6 mL of luminol/enhancer solution to 6&#160;mL of stable peroxide solution. Avoid light.&#160;</li>
			<li>Cover the entire surface of the membrane with substrate working solution and incubate for 5 min.</li>
			<li>Scan the membrane in a chemiluminescent imaging system for 1-3 s.</li>
		</ol>
		</li>
		<li>Protocol 2: Use 2x diluted commercial chemiluminescent nucleic acid detection kit and follow the steps 3.10.1.1-3.10.1.8.</li>
		<li>Protocol 3: Use self-made reagents6.
		<ol>
			<li>Prepare blocking buffer: mix 0.1 M Tris-HCl at pH 7.5, 0.1 M NaCl, 2 mM MgCl2, and 3% bovine serum albumin Fraction V. AP 7.5 buffer: mix 0.1 M Tris-HCl at pH 7.5, 0.1 M NaCl, and 2 mM MgCl2. AP 9.5 buffer: mix 0.1 M Tris-HCl at pH 9.5, 0.1 M NaCl, and 50 mM MgCl2. TE buffer: mix 10 mM Tris-HCl at pH 8.0, 1 mM EDTA at pH 8.0.</li>
			<li>Soak the membrane in blocking buffer at 30 &#176;C for 1 h.</li>
			<li>Add 8.5 &#956;L of streptavidin alkaline phosphatase to 10 mL of AP 7.5 Buffer. Shake the membrane gently in this solution at RT for 10 min. Then, wash the membrane 2x in 15 mL of AP 7.5 buffer for 10 min.</li>
			<li>Wash the membrane one more time in 20 mL of AP 9.5 buffer for 10 min. Add 20 mL of TE buffer to stop the reaction.</li>
			<li>Add 7.5 mL of development solution onto the membrane, and scan the membrane in a chemiluminescent imaging system for 1-3 s.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol><li>Bai, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sox30+initiates+transcription+of+haploid+genes+during+late+meiosis+and+spermiogenesis+in+mouse+testes%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Sox30 initiates transcription of haploid genes during late meiosis and spermiogenesis in mouse testes.</a> Development. 145 (13), (2018).</li>
<li>Watanabe, T., Lin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Posttranscriptional+regulation+of+gene+expression+by+Piwi+proteins+and+piRNAs%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">Posttranscriptional regulation of gene expression by Piwi proteins and piRNAs.</a> Molecular Cell. 56 (1), 18-27 (2014).</li>
<li>Alonso, N., Guillen, R., Chambers, J. W., Leng, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rapid+and+sensitive+high-throughput+screening+method+to+identify+compounds+targeting+protein-nucleic+acids+interactions%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">A rapid and sensitive high-throughput screening method to identify compounds targeting protein-nucleic acids interactions.</a> Nucleic Acids Research. 43 (8), 52(2015).</li>
<li>Hwang, H., Myong, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+induced+fluorescence+enhancement+%28PIFE%29+for+probing+protein-nucleic+acid+interactions%5BTitle%5D)+AND+%22Chemical+Society+Reviews%22%5BJournal%5D)">Protein induced fluorescence enhancement (PIFE) for probing protein-nucleic acid interactions.</a> Chemical Society Reviews. 43 (4), 1221-1229 (2014).</li>
<li>Gustafsdottir, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+analysis+of+DNA-protein+interactions+by+proximity+ligation%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">In vitro analysis of DNA-protein interactions by proximity ligation.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (9), 3067-3072 (2007).</li>
<li>Li, Y., Jiang, Z., Chen, H., Ma, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+modified+quantitative+EMSA+and+its+application+in+the+study+of+RNA--protein+interactions%5BTitle%5D)+AND+%22Journal+of+Biochemical+and+Biophysical+Methods%22%5BJournal%5D)">A modified quantitative EMSA and its application in the study of RNA--protein interactions.</a> Journal of Biochemical and Biophysical Methods. 60 (2), 85-96 (2004).</li>
<li>Fahrer, J., Kranaster, R., Altmeyer, M., Marx, A., Burkle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+analysis+of+the+binding+affinity+of+poly%28ADP-ribose%29+to+specific+binding+proteins+as+a+function+of+chain+length%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">Quantitative analysis of the binding affinity of poly(ADP-ribose) to specific binding proteins as a function of chain length.</a> Nucleic Acids Research. 35 (21), 143(2007).</li>
<li>Hsieh, Y. W., Alqadah, A., Chuang, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+Optimized+Protocol+for+Electrophoretic+Mobility+Shift+Assay+Using+Infrared+Fluorescent+Dye-labeled+Oligonucleotides%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides.</a> Journal of Visualized Experiments. (117), (2016).</li>
<li>Yan, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Orphan+Nuclear+Receptor+Nur77+Inhibits+Cardiac+Hypertrophic+Response+to+Beta-Adrenergic+Stimulation%5BTitle%5D)+AND+%22Molecular+and+Cellular+Biology%22%5BJournal%5D)">Orphan Nuclear Receptor Nur77 Inhibits Cardiac Hypertrophic Response to Beta-Adrenergic Stimulation.</a> Molecular and Cellular Biology. 35 (19), 3312-3323 (2015).</li>
<li>Hellman, L. M., Fried, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrophoretic+mobility+shift+assay+%28EMSA%29+for+detecting+protein-nucleic+acid+interactions%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions.</a> Nature Protocols. 2 (8), 1849-1861 (2007).</li>
<li>Fillebeen, C., Wilkinson, N., Pantopoulos, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrophoretic+mobility+shift+assay+%28EMSA%29+for+the+study+of+RNA-protein+interactions%3A+the+IRE%2FIRP+example%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Electrophoretic mobility shift assay (EMSA) for the study of RNA-protein interactions: the IRE/IRP example.</a> Journal of Visualized Experiments. (94), (2014).</li>
<li>Nishida, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hierarchical+roles+of+mitochondrial+Papi+and+Zucchini+in+Bombyx+germline+piRNA+biogenesis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Hierarchical roles of mitochondrial Papi and Zucchini in Bombyx germline piRNA biogenesis.</a> Nature. 555 (7695), 260-264 (2018).</li>
<li>Anders, C., Jinek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+enzymology+of+Cas9%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">In vitro enzymology of Cas9.</a> Methods in Enzymology. 546, 1-20 (2014).</li>
<li>Zhao, H., Zheng, J., Li, Q. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+plant+in+vitro+assay+system+for+pre-mRNA+cleavage+during+3%27-end+formation%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">A novel plant in vitro assay system for pre-mRNA cleavage during 3'-end formation.</a> Plant Physiology. 157 (3), 1546-1554 (2011).</li>
<li>Vourekas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+RNA+helicase+MOV10L1+binds+piRNA+precursors+to+initiate+piRNA+processing%5BTitle%5D)+AND+%22Genes+%26+Development%22%5BJournal%5D)">The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing.</a> Genes & Development. 29 (6), 617-629 (2015).</li>
<li>Gregersen, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MOV10+Is+a+5%27+to+3%27+RNA+helicase+contributing+to+UPF1+mRNA+target+degradation+by+translocation+along+3%27+UTRs%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">MOV10 Is a 5' to 3' RNA helicase contributing to UPF1 mRNA target degradation by translocation along 3' UTRs.</a> Molecular Cell. 54 (4), (2014).</li>
<li>Talwar, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+DEAD-box+protein+DDX43+%28HAGE%29+is+a+dual+RNA-DNA+helicase+and+has+a+K-homology+domain+required+for+full+nucleic+acid+unwinding+activity%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">The DEAD-box protein DDX43 (HAGE) is a dual RNA-DNA helicase and has a K-homology domain required for full nucleic acid unwinding activity.</a> The Journal of Biological Chemistry. 292 (25), 10429-10443 (2017).</li>
<li>Nagy, N. M., Konya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Study+of+fast+and+slow+consecutive+processes+by+heterogeneous+isotope+exchange+using+P-32+radiotracer%5BTitle%5D)+AND+%22Journal+of+Radioanalytical+And+Nuclear+Chemistry%22%5BJournal%5D)">Study of fast and slow consecutive processes by heterogeneous isotope exchange using P-32 radiotracer.</a> Journal of Radioanalytical And Nuclear Chemistry. 318 (3), 2349-2353 (2018).</li>
<li>Wilson, D. L., Beharry, A. A., Srivastava, A., O'Connor, T. R., Kool, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence+Probes+for+ALKBH2+Allow+the+Measurement+of+DNA+Alkylation+Repair+and+Drug+Resistance+Responses%5BTitle%5D)+AND+%22Angewandte+Chemie%22%5BJournal%5D)">Fluorescence Probes for ALKBH2 Allow the Measurement of DNA Alkylation Repair and Drug Resistance Responses.</a> Angewandte Chemie. 57 (39), 12896-12900 (2018).</li>
<li>Wilchek, M., Bayer, E. A., Livnah, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Essentials+of+biorecognition%3A+the+%28strept%29avidin-biotin+system+as+a+model+for+protein-protein+and+protein-ligand+interaction%5BTitle%5D)+AND+%22Immunology+Letters%22%5BJournal%5D)">Essentials of biorecognition: the (strept)avidin-biotin system as a model for protein-protein and protein-ligand interaction.</a> Immunology Letters. 103 (1), 27-32 (2006).</li>
<li>Trippier, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthetic+strategies+for+the+biotinylation+of+bioactive+small+molecules%5BTitle%5D)+AND+%22ChemMedChem%22%5BJournal%5D)">Synthetic strategies for the biotinylation of bioactive small molecules.</a> ChemMedChem. 8 (2), 190-203 (2013).</li>
<li>Rodgers, J. T., Patel, P., Hennes, J. L., Bolognia, S. L., Mascotti, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+biotin-labeled+nucleic+acids+for+protein+purification+and+agarose-based+chemiluminescent+electromobility+shift+assays%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">Use of biotin-labeled nucleic acids for protein purification and agarose-based chemiluminescent electromobility shift assays.</a> Analytical Biochemistry. 277 (2), 254-259 (2000).</li>
<li>Panda, A. C., Martindale, J. L., Gorospe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Affinity+Pulldown+of+Biotinylated+RNA+for+Detection+of+Protein-RNA+Complexes%5BTitle%5D)+AND+%22Bio-Protocol%22%5BJournal%5D)">Affinity Pulldown of Biotinylated RNA for Detection of Protein-RNA Complexes.</a> Bio-Protocol. 6 (24), (2016).</li>
<li>Bednarek, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((mRNAs+biotinylated+within+the+5%27+cap+and+protected+against+decapping%3A+new+tools+to+capture+RNA+-+protein+complexes%5BTitle%5D)+AND+%22Philosophical+Transactions+Of+the+Royal+Society+B-Biological+Sciences%22%5BJournal%5D)">mRNAs biotinylated within the 5' cap and protected against decapping: new tools to capture RNA - protein complexes.</a> Philosophical Transactions Of the Royal Society B-Biological Sciences. 373 (1762), (2018).</li>
<li>Souquet, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MEIOB+Targets+Single-Strand+DNA+and+Is+Necessary+for+Meiotic+Recombination%5BTitle%5D)+AND+%22Plos+Genetics%22%5BJournal%5D)">MEIOB Targets Single-Strand DNA and Is Necessary for Meiotic Recombination.</a> Plos Genetics. 9 (9), (2013).</li>
<li>Luo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MEIOB+exhibits+single-stranded+DNA-binding+and+exonuclease+activities+and+is+essential+for+meiotic+recombination%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">MEIOB exhibits single-stranded DNA-binding and exonuclease activities and is essential for meiotic recombination.</a> Nature Communications. 4, 2788(2013).</li>
<li>Xu, Y., Greenberg, R. A., Schonbrunn, E., Wang, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Meiosis-specific+proteins+MEIOB+and+SPATA22+cooperatively+associate+with+the+single-stranded+DNA-binding+replication+protein+A+complex+and+DNA+double-strand+breaks%5BTitle%5D)+AND+%22Biology+of+Reproduction%22%5BJournal%5D)">Meiosis-specific proteins MEIOB and SPATA22 cooperatively associate with the single-stranded DNA-binding replication protein A complex and DNA double-strand breaks.</a> Biology of Reproduction. 96 (5), 1096-1104 (2017).</li>
</ol>

No conflicts of interest are declared.

Investigating protein-nucleic acid interactions is critical to our understanding of molecular mechanisms underlying diverse biological processes. For example, MEIOB is a testis-specific protein essential for meiosis and fertility in mammals25,26,27. MEIOB contains an OB domain that binds to single-stranded DNA and exhibits 3&#39;&#160;to 5&#39;&#160;exonuclease activity26, which directly relates to its ph...

Protein-nucleic acid interactions are involved in many essential cellular processes such as DNA repair, replication, transcription, RNA processing, and translation. Protein interactions with specific DNA sequences within the chromatin are required for the tight control of gene expression at the transcriptional level1. Precise posttranscriptional regulation of numerous coding and noncoding RNAs necessitates extensive and complicated interactions between any protein and RNA2. These layers of gene expression regulatory mechanism comprise a cascade of dynamic intermolecular events, which are coordinated by interactions of transcription/epigenetic factors or RNA-binding proteins with their nucleic acid targets, as well as protein-protein interactions. To dissect whether proteins in vivo are directly or indirectly associated with nucleic acids and how such associations occur and culminate, in vitro biochemical assays are conducted to examine the binding affinity or enzymatic activity of proteins of interest on designed substrates of DNA and/or RNA.
Many techniques have been developed to detect and characterize nucleic acid-protein complexes, including the electrophoretic mobility shift assay (EMSA), also termed gel retardation assay or band shift assay3,4,5. EMSA is a versatile and sensitive biochemical method that is widely used for studying the direct binding of proteins with nucleic acids. EMSA relies on gel electrophoretic shift in bands, which are routinely visualized using chemiluminescence to detect biotin labels6,7, the fluorescence of fluorophore labels8,9, or by autoradiography of radioactive 32P labels10,11. Other purposes of biochemical studies are the identification and characterization of nucleic acid-processing activity of proteins, such as &#160;nuclease-based reactions to assess the cleavage products from nucleic acid substrates12,13,14 and DNA/RNA structure-unwinding assays to evaluate helicase activities15,16,17.
In such enzymatic activity assays, the radioisotope-labeled or fluorophore-labeled nucleic acids are often used as substrates due to their high sensitivity. Analysis of radiographs of enzymatic reactions involving 32P labeled radiotracers has been found to be sensitive and reproducible18. Yet, in an increasing number of laboratories in the world, the usage of radioisotopes is restricted or even prohibited due to the health risks associated with potential exposure. In addition to biosafety concerns, other drawbacks are the required necessary equipment to conduct work with radioisotopes, required radioactivity license, short half-life of 32P (about 14 days), and gradual deterioration of the probe quality due to radiolysis. Thus, alternative non-isotopic methods have been developed (i.e., labeling the probe with fluorophores enables detection by fluorescent imaging19). However, a high-resolution imaging system is needed when using fluorescently labeled probes. Biotin, a commonly used label, readily binds to biological macromolecules such as proteins and nucleic acids. Biotin-streptavidin system operates efficiently and improves detection sensitivity without increasing non-specific background20,21. Besides EMSA, biotin is widely used for protein purification and RNA pull-down, among others22,23,24.
This protocol successfully uses biotin-labeled nucleic acids as substrates for in vitro biochemical assays that include EMSA, in addition to enzymatic reactions in which biotin has not been commonly used. The MEIOB OB domain is constructed and two mutants (truncation and point mutation) are expressed as GST fusion proteins25,26,27, as well as mouse MOV10 recombinant FLAG fusion protein16. This report highlights the effectiveness of this combined protocol for protein purification and biotin-labeled assays for miscellaneous experimental purposes.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf, Germany</td><td>5242R</td><td></td></tr><tr><td>Chemiluminescent Imaging System</td><td>Tanon, China</td><td>5200</td><td></td></tr><tr><td>Digital sonifer</td><td>Branson, USA</td><td>BBV12081048A</td><td>450 Watts; 50/60 HZ</td></tr><tr><td>Semi-dry electrophoretic blotter</td><td>Hoefer, USA</td><td>TE77XP</td><td></td></tr><tr><td>Tube Revolver&amp;nbsp;</td><td>Crystal, USA</td><td>3406051</td><td></td></tr><tr><td>UV-light cross-linker</td><td>UVP, USA</td><td>CL-1000</td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Amicon Ultra-4 Centrifugal Filter&amp;nbsp;</td><td>Milipore, USA</td><td>UFC801096</td><td>4 ml/10 K</td></tr><tr><td>Nylon membrane</td><td>Thermo Scientific, USA</td><td>TG263940A</td><td></td></tr><tr><td>TC-treated Culture Dish</td><td>Corning, USA</td><td>430167</td><td>100 mm&amp;nbsp;</td></tr><tr><td>TC-treated Culture Dish</td><td>Corning, USA</td><td>430597</td><td>150 mm&amp;nbsp;</td></tr><tr><td>Microtubes tubes</td><td>AXYGEN, USA</td><td>MCT-150-C</td><td>1.5 mL&amp;nbsp;</td></tr><tr><td>Tubes</td><td>Corning, USA</td><td>430791</td><td>15 mL</td></tr><tr><td>&lt;strong&gt;Reagents&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ampicillin</td><td>SunShine Bio, China</td><td>8h288h28</td><td></td></tr><tr><td>Anti-FLAG M2 magnetic beads</td><td>Sigma, USA</td><td>M8823</td><td></td></tr><tr><td>ATP</td><td>Thermo Scientific, USA</td><td>591136</td><td></td></tr><tr><td>BCIP/NBT Alkaline Phosphatase Color Development Kit</td><td>Beyotime, China</td><td>C3206</td><td></td></tr><tr><td>CelLytic&lt;sup&gt;TM&lt;/sup&gt; M Cell Lysis Reagent&amp;nbsp;</td><td>Sigma, USA</td><td>107M4071V</td><td></td></tr><tr><td>ClonExpress II one step cloning kit&amp;nbsp;</td><td>Vazyme, China</td><td>C112</td><td></td></tr><tr><td>Chemiluminescent Nucleic Acid Detection Kit</td><td>Thermo Scientific, USA</td><td>T1269950</td><td></td></tr><tr><td>dNTP</td><td>Sigma-Aldrich, USA</td><td>DNTP100-1KT</td><td></td></tr><tr><td>DMEM</td><td>Gibco, USA</td><td>10569044</td><td></td></tr><tr><td>DPBS buffer</td><td>Gibco, USA</td><td>14190-136</td><td></td></tr><tr><td>EDTA</td><td>Invitrogen, USA</td><td>AM9260G</td><td>0.5 M</td></tr><tr><td>EDTA free protease inhibitor cocktail</td><td>Roche, USA</td><td>04693132001</td><td></td></tr><tr><td>EndoFree Maxi Plasmid Kit&amp;nbsp;</td><td>Vazyme, China</td><td>&amp;nbsp;&lt;br/&gt; DC202</td><td></td></tr><tr><td>FastPure Gel DNA Extraction Mini Kit</td><td>Vazyme, China</td><td>DC301-01</td><td></td></tr><tr><td>FBS</td><td>Gibco, USA</td><td>10437028</td><td></td></tr><tr><td>FLAG peptide</td><td>Sigma, USA</td><td>F4799</td><td></td></tr><tr><td>Glycerol</td><td>Sigma, USA</td><td>SHBK3676</td><td></td></tr><tr><td>GST Bulk Kit</td><td>GE Healthcare, USA</td><td>27-4570-01</td><td></td></tr><tr><td>HEPES buffer</td><td>Sigma, USA</td><td>SLBZ2837</td><td>1 M&amp;nbsp;</td></tr><tr><td>IPTG</td><td>Thermo Scientific, USA</td><td>34060</td><td></td></tr><tr><td>KoAc</td><td>Sangon Biotech, China</td><td>127-08-02</td><td></td></tr><tr><td>Lipofectamin 3000 Transfection Reagent</td><td>Thermo Scientific, USA</td><td>L3000001</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Invitrogen, USA</td><td>AM9530G</td><td>1 M</td></tr><tr><td>NaCl</td><td>Invitrogen, USA</td><td>AM9759&lt;br/&gt; &amp;nbsp;</td><td>5 M&amp;nbsp;</td></tr><tr><td>NP-40</td><td>Amresco, USA</td><td>M158-500ML</td><td></td></tr><tr><td>Opti-MEM medium</td><td>Gibco, USA</td><td>31985062</td><td></td></tr><tr><td>PBS</td><td>Gibco, USA</td><td>10010023</td><td>PH 7.4</td></tr><tr><td>RNase Inhibitor</td><td>Promega, USA</td><td>N251B</td><td></td></tr><tr><td>Streptavidin alkaline phosphatase</td><td>Promega, USA</td><td>V5591</td><td></td></tr><tr><td>TBE</td><td>Invitrogen, USA</td><td>15581044</td><td></td></tr><tr><td>Tris-HCI Buffer&amp;nbsp;</td><td>Invitrogen, USA</td><td>15567027</td><td>1 M, PH 7.4</td></tr><tr><td>Tris-HCI Buffer&amp;nbsp;</td><td>Invitrogen, USA</td><td>15568025</td><td>1 M, PH 8.0</td></tr><tr><td>Tween-20</td><td>Sangon Biotech, China</td><td>A600560</td><td></td></tr></tbody></table>

in vitro biochemical assays using biotin labels to study protein-nucleic acid interactions

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

The protein structure of MEIOB and the expression constructs used in this study are illustrated in Figure 1A. OB folds in MEIOB are compact barrel-like structures that can recognize and interact with single-stranded nucleic acids. One of the OB domains (aa 136-307, construct A) binds single stranded DNA (ssDNA), the truncated protein (aa 136-178 truncations, construct C) and the point mutant form (R235A mutation, construct E) of MEIOB do not have DNA-binding activity<sup cla...

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

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Research

JoVE Journal

Genetics

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein.  This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

This video demonstrates a technique that uses biotinylated cell-penetrating peptides to detect intracellular protein-protein interactions. The cell-penetrating peptides facilitate the conjugated peptide to enter a cell and specifically bind to its interacting proteins. The biotin, after binding to avidin, allows purification of the interacting proteins’ complex from a cell lysate.

Biotinylated Cell-Penetrating Peptide Probe to Detect Protein-Protein Interactions

In this video, we demonstrate the procedure to determine the helicase activity of a target protein to unwind the biotin-labeled dsRNA substrate. The activity of the enzyme was identified by analyzing the electrophoretic mobility shift, followed by a chemiluminescence assay using chemiluminescent enzyme-conjugated streptavidin.

Helicase Activity Measurement of a Target Protein Using Biotin-Labeled RNA Duplexes

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA.  High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its ε subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with ε’s inhibitory C-terminal domain.

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis.

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

This article describes a protocol for studying DNA-protein interactions using a streptavidin-based biolayer interferometry (BLI) system. It outlines the essential steps and considerations for utilizing either basic or advanced binding kinetics to determine the equilibrium binding affinity (KD) of the interaction.

Analyzing DNA-Protein Interactions with Streptavidin-Based Biolayer Interferometry

The protocol presents a method for in vivo covalent attachment of biotin to proteins based on their proximity to a biotin ligase fused to a protein of interest. This modification allows for a selective enrichment of the proteins using streptavidin beads as needed in protein interaction studies.

In Vivo Proximity Biotinylation for Protein Interaction Studies in Paramecium tetraurelia

Learn biotin-labeled biochemical assays for studying protein-nucleic acid interactions. This robust, efficient method enables reproducible experiments without special equipment and can be completed within a single day.

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

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