Name: Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80 (Video) | JoVE Journal
Uploaded: 2015-05-03T14:00:00.000+00:00
Duration: 12 min 28 s
Description: Learn a protein purification method to detect sumoylation and ubiquitination of kinetochore proteins Ndc10 and Ndc80 in Saccharomyces cerevisiae using western blot analysis.

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5 minutes

We thank Dr. Oliver Kerscher for support and advice and members of the Basrai laboratory for their support and comments on the paper. This work was supported by the National Institutes of Health Intramural Research Program.

1. Growth of Yeast Cells
<ol>
	<li>Inoculate yeast cells in 30 ml of YPD (Table 1) in a small flask. Incubate at 30 &#176;C overnight with shaking.</li>
	<li>Dilute the cells to an optical density of 0.2 at 600 nm (OD600 = 0.2) in 50 ml of YPD and incubate at 30 &#176;C with shaking.</li>
	<li>Grow the culture to an optical density of 1.0 at 600 nm (OD600 = 1.0).</li>
	<li>Centrifuge cells for 5 min at 2,000 x g and discard the supernatant.</li>
	<li>Resuspend the cell pellet in 40 ml sterile water and centrifuge for 5 min at 2,000 x g to wash the cells. Discard the supernatant and store the cell pellet at -20 &#176;C.</li>
</ol>
2. Extraction of Proteins
<ol>
	<li>Resuspend cells in 0.5 ml of ice-cold guanidine buffer (Table 1) for pull-down assay using Ni-NTA superflow beads or buffer A (Table 1) for immunoprecipitation. Keep tubes on ice at all times.</li>
	<li>Transfer to 2 ml screw cap tube.</li>
	<li>Add the same volume of glass beads (Table of Materials).</li>
	<li>Bead-beat the cells in a mini bead beater (Table of Materials) for 2 min at room temperature, then place on ice for 2-3 min. Repeat this three times.</li>
	<li>Vortex on high speed at 4 &#176;C for 30-60 min. Check the cells by visualization under the microscope to ensure that the cells are lysed. 
	Note: Lysed cells will appear as dark ghosts and lack a boundary or defined shape. Optimally at least 80% of the cells should be lysed.</li>
	<li>Puncture a hole in the bottom of the tube using a push pin and place in a collection 15 ml conical tube (screw cap should be loose).</li>
	<li>Centrifuge at 1,000 x g for 1 min to collect the lysate.</li>
	<li>Transfer the lysate to a micro centrifuge tube.</li>
	<li>Centrifuge at 15,000 x g for 30 min at 4 &#176;C to collect the extracted proteins.</li>
	<li>Measure the concentration of the extracted proteins using the protein assay kit (Table of Materials) and normalize all extracts to contain the same amount of protein. Bring the total volume to 1 ml with appropriate buffer. 
	Note: Around 5 mg of total protein is obtained from 50 OD600 cells.</li>
	<li>Save 50 &#181;l (250 &#181;g protein if the total protein extracted from step 2.10 is 5 mg) as whole cell extract (WCE). Add 50 &#181;l of 2x Laemmli sample buffer (Table 1) and incubate at 100 &#176;C in a heat block for 3-5 min. Load 10 &#181;l of each sample in a SDS-PAGE gel for Western Blot analysis (Section 5: Western blot analysis).</li>
</ol>
3. Purification of HF-Smt3 Conjugates
<ol>
	<li>Obtain the Ni-NTA superflow beads (Table of Materials) necessary for the experiments (100 &#181;l of beads per sample) by low speed centrifugation (800-1,500 x g) for 1 min. Remove the supernatant.</li>
	<li>Wash beads 5x with 1 ml of PBS (Table of Materials): Invert top-over-bottom until the beads are resuspended, collect beads by low speed centrifugation, and remove the supernatant.</li>
	<li>Suspend beads in 1 ml of guanidine buffer (Table 1) and aliquot them into the number of tubes corresponding to samples to be processed. Collect beads by low speed centrifugation, and remove the supernatant. Mix the beads well by inverting top-over bottom. 
	Note: Mix beads well by inverting top-over bottom.</li>
	<li>For each sample, add 950 &#181;l of the remaining WCE (from Step 2.11: Extraction of proteins) to 100 &#181;l of Ni-NTA superflow beads.</li>
	<li>Incubate on a rocking platform at 4 &#176;C for at least 4 hr or overnight.</li>
	<li>Centrifuge at 800-1,500 x g for 1 min at 4 &#176;C.</li>
	<li>Save 50 &#181;l as supernatant (SUP). Add 50 &#181;l of 2x Laemmli sample buffer and incubate at 100 &#176;C in a heat block for 3-5 min. Load 10 &#181;l of each sample in an SDS-PAGE gel for Western Blot analysis (Section 5: Western blot analysis).</li>
	<li>Wash beads once with 1 ml of guanidine buffer for 5-10 min on a rocking platform.</li>
	<li>Wash beads 5x with 1 ml of breaking buffer (Table 1) for 5-10 min on a rocking platform.</li>
	<li>Resuspend beads in 90 &#181;l of 2x Laemmli sample buffer.</li>
	<li>Add 10 &#181;l of 1 M imidazole. 
	Note: Imidazole helps to dissociate the His-tagged protein from the Ni-NTA beads due to competing interaction between imidazole and the beads.</li>
	<li>Incubate at 100 &#176;C in a heat block for 3-5 min.</li>
	<li>Vortex, then centrifuge at 13,000 x g for 30 sec. Transfer the supernatant to a fresh tube.</li>
	<li>Load 10-20 &#181;l of each sample in an SDS-PAGE gel for Western Blot analysis (Section 5: Western blot analysis). 
	Note: 10-20 &#181;l corresponds to 0.5-1.0 mg of input, if 5 mg of WCE is used.</li>
</ol>
4. Immunoprecipitation of Myc-tagged Kinetochore Proteins
<ol>
	<li>Obtain anti-c-Myc agarose affinity gel antibody (Table of Materials) necessary for the experiments (25 &#181;l of resin per sample) by low speed centrifugation (800-1,500 x g). Remove the supernatant.</li>
	<li>Wash resin 5x with 1 ml of buffer A: Invert top-over-bottom until the resin is resuspended, collect resin by low speed centrifugation, and remove the supernatant.</li>
	<li>Suspend resin in 1 ml of buffer A and aliquot them into the number of tubes corresponding to samples to be processed. Collect resin by low speed centrifugation, and remove the supernatant. 
	Note: Mix the resin well by inverting top-over-bottom.</li>
	<li>Add 950 &#181;l of the remaining WCE (from Step 2.11: Extraction of proteins) to 25 &#181;l of resin.</li>
	<li>Incubate on a rocking platform at 4 &#176;C overnight.</li>
	<li>Centrifuge at 800-1,500 x g for 1 min at 4 &#176;C.</li>
	<li>Save 50 &#181;l as supernatant (SUP). Add 50 &#181;l of 2x Laemmli sample buffer and incubate at 100 &#176;C in a heat block for 3-5 min. Load 10 &#181;l of each sample in an SDS-PAGE gel for Western Blot analysis (Section 5: Western blot analysis).</li>
	<li>Wash resin 5x with 1 ml of buffer A: Invert top-over-bottom until the resin is resuspended, collect resin by low speed centrifugation, and remove the supernatant.</li>
	<li>Resuspend the resin in 100 &#181;l of SUMEB sample buffer (Table 1). 
	Note: SUMEB sample buffer contains 8 M Urea and 1 % SDS (strong denaturing condition).</li>
	<li>Incubate at 100 &#176;C in a heat block for 3-5 min.</li>
	<li>Vortex, then centrifuge at 13,000 x g for 30 sec. Transfer the supernatant to a fresh tube.</li>
	<li>Load 10-20 &#181;l of each sample in an SDS-PAGE gel for Western Blot analysis (Section 5: Western blot analysis). 
	Note: 10-20 &#181;l corresponds to 0.5-1.0 mg of input, if 5 mg of WCE is used.</li>
</ol>
5. Western Blot Analysis
<ol>
	<li>Load the protein extracts on 4-12% Bis-Tris gels (Table of Materials) and perform electrophoresis at 120 V for 90 min (Running buffer: Table of Materials).</li>
	<li>Transfer the proteins from gel to a nitrocellulose membrane (Table of Materials) using a transfer apparatus at 30 V for 90 min (Transfer buffer: Table of Materials).</li>
	<li>Block the membrane in 5% milk/1x TBST (Table1) for 1 hr at room temperature.</li>
	<li>Incubate membrane with primary antibody (Table of Materials) in 5% milk/1x TBST overnight at 4 &#176;C. For primary antibodies, use a dilution of 1:1,000 (anti-Flag and anti-Ub antibodies) or 1:5,000 (anti-Myc antibody).</li>
	<li>Wash membrane 3x for 10 min each with 1x TBST.</li>
	<li>Incubate membrane with secondary antibody (Table of Materials) in 5% milk/1x TBST for 1 hr at room temperature. Use a 1:5,000 dilution of secondary antibodies.</li>
	<li>Wash membrane 3x for 10 min each with 1x TBST.</li>
	<li>Incubate membrane with ECL working solution (Table of Materials) for 5 min.</li>
	<li>Expose the membrane to blue sensitive X-Ray film (Table of Materials) and develop using an automatic developer (Table of Materials).</li>
</ol>

<ol><li>Johnson, P. R., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SUMO-1%3A+Ubiquitin+gains+weight%5BTitle%5D)+AND+%22Trends+Cell+Biol%22%5BJournal%5D)">SUMO-1: Ubiquitin gains weight.</a> Trends Cell Biol. 7, 408-413 (1997).</li>
<li>Hewawasam, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Psh1+is+an+E3+ubiquitin+ligase+that+targets+the+centromeric+histone+variant+Cse4%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">Psh1 is an E3 ubiquitin ligase that targets the centromeric histone variant Cse4.</a> Mol Cell. 40, 444-454 (2010).</li>
<li>Ranjitkar, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+E3+ubiquitin+ligase+prevents+ectopic+localization+of+the+centromeric+histone+H3+variant+via+the+centromere+targeting+domain%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">An E3 ubiquitin ligase prevents ectopic localization of the centromeric histone H3 variant via the centromere targeting domain.</a> Mol Cell. 40, 455-464 (2010).</li>
<li>Au, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+role+of+the+N+terminus+of+budding+yeast+histone+H3+variant+Cse4+in+ubiquitin-mediated+proteolysis%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">A novel role of the N terminus of budding yeast histone H3 variant Cse4 in ubiquitin-mediated proteolysis.</a> Genetics. 194, 513-518 (2013).</li>
<li>Akiyoshi, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Mub1%2FUbr2+ubiquitin+ligase+complex+regulates+the+conserved+Dsn1+kinetochore+protein%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">The Mub1/Ubr2 ubiquitin ligase complex regulates the conserved Dsn1 kinetochore protein.</a> PLoS Genet. 9, e1003216(2013).</li>
<li>Biggins, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+conserved+protein+kinase+Ipl1+regulates+microtubule+binding+to+kinetochores+in+budding+yeast%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">The conserved protein kinase Ipl1 regulates microtubule binding to kinetochores in budding yeast.</a> Genes Dev. 13, 532-544 (1999).</li>
<li>Cheeseman, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phospho-regulation+of+kinetochore-microtubule+attachments+by+the+Aurora+kinase+Ipl1p%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Phospho-regulation of kinetochore-microtubule attachments by the Aurora kinase Ipl1p.</a> Cell. 111, 163-172 (2002).</li>
<li>Liu, D., Lampson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+kinetochore-microtubule+attachments+by+Aurora+B+kinase%5BTitle%5D)+AND+%22Biochem+Soc+Trans%22%5BJournal%5D)">Regulation of kinetochore-microtubule attachments by Aurora B kinase.</a> Biochem Soc Trans. 37, 976-980 (2009).</li>
<li>Montpetit, B., Hazbun, T. R., Fields, S., Hieter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sumoylation+of+the+budding+yeast+kinetochore+protein+Ndc10+is+required+for+Ndc10+spindle+localization+and+regulation+of+anaphase+spindle+elongation%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Sumoylation of the budding yeast kinetochore protein Ndc10 is required for Ndc10 spindle localization and regulation of anaphase spindle elongation.</a> J Cell Biol. 174, 653-663 (2006).</li>
<li>Furth, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exposure+of+bipartite+hydrophobic+signal+triggers+nuclear+quality+control+of+Ndc10+at+the+endoplasmic+reticulum%2Fnuclear+envelope%5BTitle%5D)+AND+%22Mol+Biol+Cell%22%5BJournal%5D)">Exposure of bipartite hydrophobic signal triggers nuclear quality control of Ndc10 at the endoplasmic reticulum/nuclear envelope.</a> Mol Biol Cell. 22, 4726-4739 (2011).</li>
<li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anaphase-promoting+complex%2Fcyclosome+controls+HEC1+stability%5BTitle%5D)+AND+%22Cell+Prolif%22%5BJournal%5D)">Anaphase-promoting complex/cyclosome controls HEC1 stability.</a> Cell Prolif. 44, 1-9 (2011).</li>
<li>Takahashi, Y., Yong-Gonzalez, V., Kikuchi, Y., Strunnikov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SIZ1%2FSIZ2+control+of+chromosome+transmission+fidelity+is+mediated+by+the+sumoylation+of+topoisomerase+II%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">SIZ1/SIZ2 control of chromosome transmission fidelity is mediated by the sumoylation of topoisomerase II.</a> Genetics. 172, 783-794 (2006).</li>
<li>Liu, C., Apodaca, J., Davis, L. E., Rao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteasome+inhibition+in+wild-type+yeast+Saccharomyces+cerevisiae+cells%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">Proteasome inhibition in wild-type yeast Saccharomyces cerevisiae cells.</a> Biotechniques. 42, 158(2007).</li>
<li>Kitamura, E., Tanaka, K., Kitamura, Y., Tanaka, T. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetochore+microtubule+interaction+during+S+phase+in+Saccharomyces+cerevisiae%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">Kinetochore microtubule interaction during S phase in Saccharomyces cerevisiae.</a> Genes Dev. 21, 3319-3330 (2007).</li>
<li>Gascoigne, K. E., Cheeseman, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetochore+assembly%3A+if+you+build+it%2C+they+will+come%5BTitle%5D)+AND+%22Curr+Opin+Cell+Biol%22%5BJournal%5D)">Kinetochore assembly: if you build it, they will come.</a> Curr Opin Cell Biol. 23, 102-108 (2011).</li>
</ol>

The authors have nothing to disclose.

Epitope tags such as HA, Myc, Flag, and GST are widely used for biochemical analysis of proteins. Construction of strains with HF-Smt3 and Myc-tagged kinetochore proteins, such as Ndc10 and Ndc80, facilitates the detection of PTMs such as sumoylation and ubiquitination. HF-Smt3 pull down assay allows the detection of sumoylated kinetochore proteins, Ndc10 and Ndc80 (Figure 1). The affinity purification protocol and western blot analysis using anti-Flag antibody establish the specificity of interaction be...

Ubiquitination and sumoylation allow the conjugation of ubiquitin and Small Ubiquitin-like MOdifier (SUMO; Smt3 in S. cerevisiae 1) to a target protein, respectively. PTMs of kinetochore proteins affect their cellular levels and protein-protein interactions during different cell cycle phases to ensure faithful chromosome segregation. For example, cellular levels of Cse4/CENP-A and outer kinetochore protein Dsn1 are regulated by ubiquitin-mediated proteolysis for ensuring genome stability 2-5. Destabilization of incorrect kinetochore-microtubule attachments requires the Ipl1/Aurora B kinase, which phosphorylates Dam1 and Ndc80 complexes that directly interact with microtubules 6-8. Despite the identification of over seventy kinetochore proteins, there are very few studies that investigate the modifications of these proteins with PTMs, e.g., ubiquitin and SUMO. A major limitation is the ability to preserve the PTMs during purification and the paucity of custom antibodies for detection of PTMs such as sumoylation, phosphorylation, methylation, and others. Characterization of sumoylated kinetochore proteins Ndc10, Cep3, Bir1, and Ndc80 utilized a custom antibody 9. Additionally, Ndc10 has been implicated as a substrate for ubiquitination 10. Human Hec1 (Ndc80 in S. cerevisiae) is also substrate for ubiquitination, regulated by APC/C-hCdh1 E3 ligase 11. Therefore, Ndc10 and Ndc80 are good candidates for optimization of the protocol to detect both sumoylation and ubiquitination in S. cerevisiae.
To facilitate the identification of sumoylation, we constructed strains that express HF-Smt3 and Myc-tagged Ndc10 or Ndc80. The use of epitope tags (HF: His6-Flag) minimizes the background due to cross-reactivity that is frequently observed in polyclonal serum raised against a candidate protein. We devised a protocol to affinity purify HF-Smt3 conjugates and then used commercial anti-Flag and anti-Myc antibodies to detect the presence of sumolyated Ndc10 and Ndc80 in the purified Smt3 preparation. For ubiquitination, we devised a modified immunoprecipitation protocol that preserves ubiquitination of the Myc-tagged kinetochore proteins and performed western blot analysis with commercial anti-Ub antibody to detect ubiquitination of Ndc10 and Ndc80.

<table><tbody><tr><td>Glass beads</td><td>BioSpec Products</td><td>11079105</td><td>0.5 mm diameter</td></tr><tr><td>Mini beadbeater</td><td>BioSpec Products</td><td>#693</td><td>8 cell disrupter</td></tr><tr><td>Ni-NTA superflow</td><td>Qiagen</td><td>30430</td><td>100 ml</td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma-Aldrich</td><td>P8215</td><td>1 ml</td></tr><tr><td>Anti-c-Myc agarose affinity gel antbody produced in rabbit</td><td>Sigma-Aldrich</td><td>A7470</td><td>1 ml</td></tr><tr><td>Monoclonal anti-Flag M2 antibody produced in mouse</td><td>Sigma-Aldrich</td><td>F1804</td><td>Primary antibody, dilution 1:1,000</td></tr><tr><td>c-Myc antibody (A-14)</td><td>Santa Cruz Biotechnology</td><td>sc-789</td><td>Primary antibody, dilution 1:5,000</td></tr><tr><td>Purified mouse antibody monoclonal 9E10</td><td>Covance</td><td>MMS-150P</td><td>Primary antibody, dilution 1:5,000</td></tr><tr><td>Ubiquitin (P4G7) monoclonal antibody</td><td>Covance</td><td>MMS-258R</td><td>Primary antibody, dilution 1:1,000</td></tr><tr><td>ECL Rabbit IgG, HRP-linked whole Ab</td><td>GE Healthcare Life Sciences</td><td>NA934V</td><td>Secondary antibody, dilution 1:5,000</td></tr><tr><td>ECL Mouse IgG, HRP-Linked Whole Ab</td><td>GE Healthcare Life Sciences</td><td>NA931V</td><td>Secondary antibody, dilution 1:5,000</td></tr><tr><td>DC protein assay</td><td>Bio-Rad</td><td>500-0116</td><td></td></tr><tr><td>Nitrocellulose membrane</td><td>Novex</td><td>LC2001</td><td>0.45 mm pore size</td></tr><tr><td>NuPAGE 4-12% Bis-Tris Protein Gels</td><td>Novex</td><td>NP0321BOX</td><td>1.0 mm, 10 well</td></tr><tr><td>NuPAGE MES SDS Running Buffer</td><td>Novex</td><td>NP0002</td><td>20x</td></tr><tr><td>NuPAGE Transfer Buffer</td><td>Novex</td><td>NP0006-1</td><td>20x</td></tr><tr><td>SuperSignal West Pico Chemiluminescent Substrate</td><td>Thermo Scientific</td><td>34078</td><td></td></tr><tr><td>10x PBS pH7.4</td><td>GIBCO</td><td>70011-044</td><td></td></tr><tr><td>Blue sensitive X-Ray film</td><td>Dbio</td><td>DBOF30003</td><td></td></tr><tr><td>Automatic developer</td><td>Kodak</td><td>M35AX-OMAT</td><td></td></tr><tr><td>Nocodazole</td><td>Sigma-Aldrich</td><td>M1404</td><td>50 mg</td></tr><tr><td>MG-132</td><td>Selleck Chemicals</td><td>S2619</td><td>25 mg</td></tr></tbody></table>

protein purification technique that allows detection of sumoylation and ubiquitination of budding yeast kinetochore proteins ndc10 and ndc80

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

To detect sumoylation of kinetochore proteins Ndc80 and Ndc10, strains with HF-Smt3 and Myc-tagged kinetochore proteins (Ndc80 or Ndc10) were constructed (Table 2), as previously described 9,12. HF-Smt3 conjugates were affinity purified using Ni-NTA beads. Western blot analysis of purified HF-Smt3 with an anti-Flag antibody allowed detection of sumoylated forms of SUMO substrates that were absent in the control strain without HF-Smt3 (Figure 1A and 1B, left pa...

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

The overall goal of this procedure is to detect simulation and ubiquitination of budding yeast kinetic core proteins, NDC 10 and NDC 80. This is accomplished by first harvesting yeast cells that express hiss flag tagged SMT three and MT tagged NDC 10 or NDC 80, and making protein extracts from these cells. The second step is to purify hiss flag tagged SMT three conjugates for the detection of summation or to immuno precipitate mik tagg kinetic core proteins for the detection of ubiquitination.Subsequently, sum ation or ubiquitination is detected by western blot analysis. Ultimately, the presence of a laddering pattern for NDC 10 and NDC 80 confirms that these proteins are simulated and ubiquitinated. The protein purification method described allows the detection of both simulation and ubiquitination of the connico proteins ND C 10 and NDC 80 in the budding yeast sacro.My cci. The main advantage of this technique is the strain we have created has two epitope attacks, one on the test protein, and the other that allows detection of simulation. The use of these tags reduces the background due to cross reactivity that is frequently observed when polyclonal serum are used.The demonstration of this procedure will be done by Canero Uni, a research fellow in my laboratory. Proteins of interest will be extracted from yeast cell pellets prepared previously and stored at minus 20 degrees Celsius. Resus, suspend the cells in 0.5 milliliters of ice cold buffer.Use guine buffer if the extract is for a pull-down assay using nickel nitrile, tri acetic acid or nickel NTA superflow beads. Use buffer A if the extract is for immunoprecipitation. Keep the tubes on ice at all times.Transfer the cell suspensions to two milliliters. Screw cap twos to each tube. Add a volume of glass beads equal to the volume of the cell suspension bead.Beat the cells in a mini bead beater for two minutes at room temperature and then place the tubes on ice for two to three minutes. Repeat the bead beating and icing three times. Vortex on high speed at four degrees Celsius for 30 to 60 minutes.Check the cells by visualization under the microscope to ensure that the cells are lysed. Lysed cells will appear as dark ghosts and lack a boundary or defined shape. Optimally at least 80%of the cells should be lyed.Next, use a push pin to puncture a hole in the bottom of the tube. Place the screw cap on loosely and place the tube in a 15 milliliter conical tube. Centrifuge at 1000 times G for one minute.To collect the lysate, transfer the lysate to a micro centrifuge tube centrifuge at 15, 000 times G for 30 minutes at four degrees Celsius to collect the extracted proteins. After measuring the concentration of the extracted proteins using a protein assay kit, normalize all extracts so each would contain the same amount of protein. Bring the total volume to one milliliter with the appropriate buffer.Approximately five milligrams of total protein is obtained from 50 OD 600 of cells. Save 50 microliters of the extracted protein as the whole cell extract. The remaining 950 microliters of protein extract will be used later for protein purification or immunoprecipitation of MT tagged proteins.Add 50 microliters of two x laly sample buffer to the 50 microliters of whole cell extract. Incubate the samples at 100 degrees Celsius in a heat block for three to five minutes before analyzing them by western blot. Begin this procedure by preparing the nickel NTA superflow beads necessary for the purification.Obtain the amount of beads needed by low speed centrifugation for one minute. Remove the supernatant and wash the beads with PBS. Add one milliliter of PBS and invert the tube top over bottom until the beads are resuspended.Collect beads by low speed centrifugation and remove the supernatant. Wash the beads in this manner a total of five times. Suspend the beads in one milliliter of guine buffer and aliquot them into the number of tubes corresponding to the samples to be processed.Collect the beads by low speed centrifugation and remove the supernatant for each sample. Add 950 microliters of the whole cell extract. Prepared earlier to 100 microliters of the beads.Incubate on a rocking platform at four degrees Celsius for at least four hours or overnight. Centrifuge at 800 to 1500 times G for one minute at four degrees Celsius. Save 50 microliters of the supernatant.Add 50 microliters of two x laly sample buffer to this supernatant and incubate at 100 degrees Celsius in a heat block for three to five minutes. 10 microliters of each sample will later be analyzed by western blot. Wash the nickel NTA superflow beads once with one milliliter of guine buffer for five to 10 minutes on a rocking platform at room temperature.Next, wash the beads five times with one milliliter of breaking buffer after the final wash with breaking buffer resuspend the beads in 90 microliters of two x laly sample buffer. Add 10 microliters of one molar iole to help dissociate the his tagged protein from the nickel NT.A beads incubate at 100 degrees Celsius in a heat block for three to five minutes. Vortex then centrifuge at 13, 000 times G for 30 seconds.Transfer the supernatant to a fresh tube. To begin this procedure, obtain the necessary amount of anti cmic aero affinity gel antibody by low speed centrifugation. Remove the supernatant, wash the resin with buffer A Add one milliliter of buffer A and invert top over bottom until the resin is resuspended.Collect the resin by low speed centrifugation and remove the supernatant. Wash a total of five times. Suspend the resin in one milliliter of buffer A and aliquot into the number of tubes corresponding to the samples to be processed.Collect the resin by low speed centrifugation and remove the supernatant. Add 950 microliters of whole cell extract to 25 microliters of resin Incubate on a rocking platform at four degrees Celsius overnight on the following day. Centrifuge at 800 to 1500 times G for one minute at four degrees Celsius.Save 50 microliters of the supernatant. Add 50 microliters of two x lam ly sample buffer to this supernatant and incubate at 100 degrees Celsius in a heat block for three to five minutes. Wash the resin with buffer a add one milliliter of buffer, a invert top over bottom until the resin is resuspended.Collect resin by low speed centrifugation and remove the supernatant after removing the supernatant from the final wash. Resuspend the resin in 100 microliters of sume sample buffer incubate at 100 degrees Celsius in a heat block for three to five minutes. Vortex then centrifuge at 13, 000 times G for 30 seconds.Transfer the supernatant to a fresh tube. Lastly, load all the samples collected from this protocol. In an SDS page, gel for Western blot analysis, yeast strains that express poly histamine flag tagged SMT three or HF SMT three and MT tagged NDC 80 and NDC 10 were used to detect simulation of kinetico proteins.HF SMT three substrates were affinity purified using nickel beads and detected with an Anti-Flag antibody. Sum moated NDC 80 was detected in the HF SMT three substrates when probed with an anti mic antibody. The absence of modified proteins in the control strains without HF SMT three indicates the specificity of interaction between HF SMT three and its target proteins.Moreover, summation of NDC 80 and NDC 10 is reduced in NOCO desole treated cells to detect ubiquitination of NDC 80 and NDC 10. Mick tagged ND C 80 or ND C 10 was immuno precipitated and western blot analysis was performed with anti mic and anti ubiquitin antibodies analysis of whole cell extract and supernatant confirmed the expression of NDC 80 M and NDC 10 M immuno precipitated samples probed with anti MIC showed multiple high molecular weight bands suggesting that NDC 80 and NDC 10 contain post-translational modifications. A laddering pattern of immuno precipitated samples probed with anti ubiquitin showed that ND C 80 and ND C 10 are ubiquitinated.The laddering patterns of NDC 80 and NDC 10 were enhanced by treatment with a proteasome inhibitor MG 1 32. Further confirming that these represent poly ubiquitination After watching this video, you should have a good understanding of the protein purification technique that allows detection of mation and vaccination of bat NN.

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Research

JoVE Journal

Biology

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Detection of Protein Ubiquitination

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Chromatographic Purification of Highly Active Yeast Ribosomes

This video describes a method to extract and purify ubiquitinylated peptides containing remnant di-glycine peptides from a complex peptide mixture. The presented method may help in identifying original ubiquitination sites in the protein.

Immunoaffinity Based Extraction of Ubiquitinylated Peptides: A Technique to Selectively Extract Ubiquitin Tagged Peptides from Purified Peptide Fractions

This video demonstrates the in vitro method for SUMOylation of substrate proteins using a sequential enzyme cascade. Further, the SUMOylated status of the protein is identified using the electrophoresis and immunoblotting technique.

SUMOylation Assay: An In Vitro Technique to Detect the SUMOylation Status of Substrate Proteins by Immunoblotting

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes.  Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

The protocol describes a step-by-step method to purify ubiquitinated proteins from mammalian cells using the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified from cells under stringent nondenaturing and denaturing conditions.

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

Ubiquitination is a critical regulator of intracellular protein functions. Here, we describe two cell lysis methods for detecting substrate protein ubiquitination levels in mammalian cells and compare their effectiveness in analyzing ubiquitination levels. Protein overexpression in HEK293T cells was induced by transient transfection, and ubiquitinated proteins were detected by western blot analysis.

Comparative Strategies for Ubiquitination Detection in Mammalian Cell Lysates Using SMAD2/SMURF2 as a Model

Learn a protein purification method to detect sumoylation and ubiquitination of kinetochore proteins Ndc10 and Ndc80 in Saccharomyces cerevisiae using western blot analysis.

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80 (Video) | JoVE Journal

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

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