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>

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	<li>Johnson, P. R., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO-1:+Ubiquitin+gains+weight.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psh1+is+an+E3+ubiquitin+ligase+that+targets+the+centromeric+histone+variant+Cse4.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+E3+ubiquitin+ligase+prevents+ectopic+localization+of+the+centromeric+histone+H3+variant+via+the+centromere+targeting+domain.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+of+the+N+terminus+of+budding+yeast+histone+H3+variant+Cse4+in+ubiquitin-mediated+proteolysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mub1/Ubr2+ubiquitin+ligase+complex+regulates+the+conserved+Dsn1+kinetochore+protein.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+conserved+protein+kinase+Ipl1+regulates+microtubule+binding+to+kinetochores+in+budding+yeast.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospho-regulation+of+kinetochore-microtubule+attachments+by+the+Aurora+kinase+Ipl1p.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+kinetochore-microtubule+attachments+by+Aurora+B+kinase.">Regulation of kinetochore-microtubule attachments by Aurora B kinase.</a> Biochem Soc Trans. 37, 976-980 (2009).</li><li>Montpetit, B., Hazbun, T. 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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 between HF-Smt3 and target proteins, as modified proteins were not detected in the control strain without HF-Smt3. The use of the Myc tag on the protein substrates validates the presence of sumoylated Ndc10 and Ndc80 in the HF-Smt3 conjugates. Furthermore, sumoylation of both Ndc10 and Ndc80 was reduced in nocodazole treated cells (Figure 1C and 1D). Decreased sumoylation of Ndc10 in response to nocodazole treatment, but not Ndc80, was previously reported by Montpetit et al. 9. This may be due to differences in experimental protocol and use of a custom anti-SUMO antibody. Our results for sumoylation of kinetochore substrates suggest that a similar protocol can be used to investigate other candidate SUMO substrates.
To detect ubiquitination, Myc-tagged kinetochore proteins were first immunoprecipitated and the IP fractions were probed with anti-Ub antibody (Figure 2). The laddering pattern of Ndc10 and Ndc80 was increased when cells were treated with MG132 to inhibit proteasome function (Figure 2B), indicating that these proteins are substrates of the proteasome. The IP fractions failed to detect sumoylation of Ndc10 or Ndc80 in western blot analysis with anti-Flag or anti-Smt3 antibody (data not shown). This may be due to technical reasons such as levels of sumoylated substrate in the IP fraction or the interference from the epitope tags. Further optimization of the IP protocol such as concentration of salts and western blotting conditions should facilitate the detection of multiple PTMs of any protein of interest. At present, sumoylation is best detected by the HF-Smt3 pull down assay followed by western blots of the candidate SUMO substrates (Figure 1).
Several details affect the efficiency of protein purification and the ability to detect the PTMs. First, all tubes should be kept on ice to inhibit the proteases except as specified. Second, a 1:1 ratio of cell suspension to glass beads is critical to disrupt the cells by the bead beater and/or vortexer. Cell lysis should be monitored by examination of the cells under the microscope. Third, it is very important to prepare a clarified lysate for the pull down assay or IP. Short or low speed centrifugation can contribute to contamination of cell debris in the lysate and this affects the ability to detect the PTMs. Finally, vigorous shaking of the beads with a large volume of clear lysate (about 1 ml) in a 1.5 ml micro centrifuge tube ensures that the beads are fully suspended for the pull down assay or IPs. In summary, protein purification and detection techniques such as that described here provide important mechanistic insights into how PTM of kinetochore proteins affect their structure and assembly for faithful chromosome segregation 14,15.

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

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 panel). As expected, multiple forms of SUMO substrates were detected in the HF-Smt3 strain. We next determined if Ndc80 and Ndc10 are present in the purified HF-Smt3 conjugate by doing western blot analysis using an anti-Myc antibody. Multiple bands that were of higher molecular weight than that of Ndc80 or Ndc10 were clearly detected (Figure 1A and 1B, right panel). Moreover, multiple bands of both Ndc80 and Ndc10 were reduced in nocodazole treated cells (Figure 1C and 1D). These results show that protein purification and western blot analysis described here allow the detection of sumoylation of Ndc80 and Ndc10, as previously described9.
To detect ubiquitination of Ndc80 and Ndc10, Myc-tagged Ndc80 or Ndc10 was immunoprecipitated (IP) and western blot analysis was performed with anti-Myc and anti-Ub antibodies (Figure 2). Analysis of whole cell extracts (WCE) and supernatant (SUP) confirmed the expression of Ndc80-Myc and Ndc10-Myc (Figure 2A). The lower molecular weight bands on the WCE may represent degradation products. IP samples probed with anti-Myc showed multiple high molecular weight bands, suggesting that Ndc80 and Ndc10 contain PTMs. A laddering pattern of IP samples probed with anti-Ub antibody showed that Ndc80 and Ndc10 are ubiquitinated (Figure 2A, &#945;-Ub). The laddering pattern of Ndc80 and Ndc10 were enhanced by treatment with proteasome inhibitor (MG132) (Figure 2B, &#945;-Ub), further confirming that these bands represent poly-ubiqutination. The representative results reveal that both Ndc80 and Ndc10 are substrates for sumoylation and ubiquitination in S. cerevisiae and that the described protein purification methods are useful for detection of PTMs such as sumoylation and ubiquitination. According to our knowledge, this is the first report that Ndc80 in S. cerevisiae is a substrate for ubiquitination, although ubiquitin mediated proteolysis of the human homolog Hec1 has been previously published 11.
<img alt="Figure 1" src="/files/ftp_upload/52482/52482fig1.jpg" /> 
Figure 1: Purification of sumolyated substrates allows the identification of kinetochore proteins Ndc80 and Ndc10 as SUMO substrates. Proteins were extracted and HF-Smt3 conjugates were prepared and analyzed by western blot analysis after SDS-PAGE separation. (A and B) Ndc80 and Ndc10 are sumoylated. Proteins were extracted from logarithmically growing cells in YPD. Left panel: Total SUMO substrates were detected with an anti-Flag antibody. Right panel: Sumoylated Ndc80 or Ndc10 was detected in the HF-Smt3 conjugates when probed with an anti-Myc antibody. (C and D) Sumoylation of Ndc80 and Ndc10 is reduced in nocodazole treated cells. Logarithmically growing cells in YPD were treated with DMSO (-) or DMSO + 20 &#956;g/ml nocodazole (+) for 2 hr at 30 &#176;C. HF-Smt3 conjugates were analyzed by western blot analysis using anti-Myc antibody. Isogenic yeast strains used are (A and C) YPH1800 (Ndc80-Myc), YMB7862 (His-Flag-SMT3 Ndc80-Myc) and (B and D) YPH1734 (Ndc10-Myc), YMB7867 (His-Flag-SMT3 Ndc10-Myc).
<img alt="Figure 2" src="/files/ftp_upload/52482/52482fig2.jpg" /> 
Figure 2: Ubiquitination of kinetochore proteins Ndc80 and Ndc10. Extraction of protein and immunoprecipitation were performed as described in the protocol. (A) Ndc80 and Ndc10 are ubiquitinated. Proteins were extracted from logarithmically growing cells in YPD. Whole cell extract (WCE), supernatant (SUP), and the immunoprecipitated (IP) fractions were subjected to SDS-PAGE. Western blots were used to detect Myc-tagged and ubiquitinated proteins with anti-Myc and anti-Ub antibodies, respectively. (B) Ubiquitination of Ndc80 and Ndc10 is enhanced in cells treated with proteasome inhibitor MG132. Logarithmically growing cells in YPD were treated with DMSO (-) or DMSO + 50 &#181;M MG132 (+) in the presence of 0.003% of SDS for 3 hr at 30 &#176;C, as previously described 13. Western blots of IP fraction were done using anti-Myc or anti-Ub antibody. Isogenic yeast strains used are YMB7278 (His-Flag-SMT3), YMB7862 (His-Flag-SMT3 Ndc80-Myc) and YMB7867 (His-Flag-SMT3 Ndc10-Myc).
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Components</td>
		</tr>
		<tr>
			<td>YPD</td>
			<td>1 % yeast extract, 2 % bacto-peptone, 2 % glucose</td>
		</tr>
		<tr>
			<td>Guanidine buffer</td>
			<td>0.1 M Tris-HCl pH 8.0, 6 M guanidine chloride, 0.5 M NaCl</td>
		</tr>
		<tr>
			<td>Buffer A</td>
			<td>50 mM Tris-HCl pH 7.5, 50 mM NaCl, 0.2 % Triton X-100, 1x protease inhibitors</td>
		</tr>
		<tr>
			<td>Breaking buffer</td>
			<td>0.1 M Tris-HCl pH 8.0, 20 % glycerol, 1 mM PMSF</td>
		</tr>
		<tr>
			<td>SUMEB sample buffer</td>
			<td>1 % SDS, 8 M Urea, 10 mM MOPS pH 6.8, 10 mM EDTA, 0.01 % bromophenol blue</td>
		</tr>
		<tr>
			<td>2x Laemmli Sample Buffer</td>
			<td>100 mM Tris-HCl pH 6.8, 4 % SDS, 20 % glycerol, 0.2 % bromophenol blue (Before use: add b-mercaptoethanol (BME) to a final concentration of 200 mM)</td>
		</tr>
		<tr>
			<td>1x TBST</td>
			<td>137 mM Sodium Chloride, 20 mM Tris-Hcl pH 7.5, 0.1% Tween-20</td>
		</tr>
	</tbody>
</table>
Table 1: Solutions.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Table 2. Yeast strains used in this study*.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Strains</td>
			<td>Parent</td>
			<td>Genotype</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>BY4741</td>
			<td></td>
			<td>MATa his3&#8710;1 leu2&#8710;0 met15&#8710;0 ura3&#8710;0</td>
			<td>Open Biosystems</td>
		</tr>
		<tr>
			<td>BY4742</td>
			<td></td>
			<td>MATa his3&#8710;1 leu2&#8710;0 lys2&#8710;0 ura3&#8710;0</td>
			<td>Open Biosystems</td>
		</tr>
		<tr>
			<td>YMB7278</td>
			<td>BY4741/BY4742</td>
			<td>MATa his3&#8710;1 leu2&#8710;0 lys2&#8710;0 ura3&#8710;0 HF-SMT3::LEU2</td>
			<td>This study</td>
		</tr>
		<tr>
			<td>YPH1734</td>
			<td></td>
			<td>MATa ura3-52 lys2-801 ade2-101 his3&#8710;200 leu2&#8710;1 trp1&#8710;63 NDC10-13Myc::kanMX6</td>
			<td>Montpetit et al., 2006</td>
		</tr>
		<tr>
			<td>YPH1800</td>
			<td></td>
			<td>MATa ura3-52 lys2-801 ade2-101 his3&#8710;200 leu2&#8710;1 trp1&#8710;63 NDC80-13Myc::His3MX6</td>
			<td>Montpetit et al., 2006</td>
		</tr>
		<tr>
			<td>YMB7862</td>
			<td>YMB7278/YPH1800</td>
			<td>MATa his3 leu2 ura3 ade2 trp1 HF-SMT3::LEU2 NDC80-Myc::His3MX6</td>
			<td>This study</td>
		</tr>
		<tr>
			<td>YMB7867</td>
			<td>YMB7278/YPH1734</td>
			<td>MATa his3 leu2 ura3 trp1 lys2 HF-SMT3::LEU2 NDC10-Myc::kanMX6</td>
			<td>This study</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>* All yeast strains are derived from Saccharomyces cerevisiae S288C.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Yeast strains used in this study.

Watch this Scientific Journal Video about Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80 at JoVE.com

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

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 ...

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Universidad San Sebastian

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Biology

12.0K Views.  National Cancer Institute, National Institute of Health. This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

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

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Biochemistry

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. This method utilizes a recombinant modified SUMO-trapping protein fragment, kmUTAG, derived from the Ulp1 SUMO protease of the stress-tolerant budding yeast Kluyveromyces marxianus. We have adapted the properties of the kmUTAG for the purpose of studying sumoylation in a variety of model systems without the use of antibodies. For the study of SUMO, KmUTAG has several advantages when compared to antibody-based approaches. This stress-tolerant SUMO-trapping reagent is produced recombinantly, it recognizes native SUMO isoforms from many species, and unlike commercially available antibodies it shows reduced affinity for free, unconjugated SUMO. Representative results shown here include the localization of SUMO conjugates in mammalian tissue culture cells and nematode oocytes.

SUMO is an essential and highly conserved, small ubiquitin-like modifier protein. In this protocol we are describing the use of a stress-tolerant recombinant SUMO-trapping protein (kmUTAG) to visualize native, untagged SUMO conjugates and their localization in a variety of cell types.

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. ...

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

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

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.

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

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.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, including cell survival, proliferation, and differentiation. It is a reversible process that consists of a covalent attachment of ubiquitin to the substrate through a cascade reaction of at least three different enzymes, composed of E1 (Ubiquitin-activation enzyme), E2 (Ubiquitin-conjugating enzyme), and E3 (Ubiquitin-ligase enzyme). The E3 complex plays an important role in substrate recognition and ubiquitylation. Here, a protocol is described to evaluate substrate ubiquitylation in mammalian cells using transient co-transfection of a plasmid encoding the selected substrate, an E3 ubiquitin ligase, and a tagged ubiquitin. Before lysis, the transfected cells are treated with the proteasome inhibitor MG132 (carbobenzoxy-leu-leu-leucinal) to avoid substrate proteasomal degradation. Furthermore, the cell extract is submitted to small-scale immunoprecipitation (IP) to purify the polyubiquitylated substrate for subsequent detection by western blotting (WB) using specific antibodies for ubiquitin tag. Hence, a consistent and uncomplicated protocol for ubiquitylation assay in mammalian cells is described to assist scientists in addressing ubiquitylation of specific substrates and E3 ubiquitin ligases.

We provide a detailed protocol for a ubiquitylation assay of a specific substrate and an E3 ubiquitin-ligase in mammalian cells. HEK293T cell lines were used for protein overexpression, the polyubiquitylated substrate was purified from cell lysates by immunoprecipitation, and resolved in SDS-PAGE. Immunoblotting was used to visualize this post-translational modification.

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, ...

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate protein. The ubiquitination process occurs intracellularly in eukaryotes and regulates almost all basic cellular biological processes. Purification of ubiquitinated proteins aids the investigation of the role of ubiquitination in controlling the function of substrate proteins. Here, a step-by-step procedure to purify ubiquitinated proteins in mammalian cells is described with the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified under stringent nondenaturing and denaturing conditions. Total cellular Flag-tagged p53 protein was purified with anti-Flag antibody-conjugated agarose under nondenaturing conditions. Alternatively, total cellular His-tagged ubiquitinated protein was purified using nickel-charged resin under denaturing conditions. Ubiquitinated p53 proteins in the eluates were successfully detected with specific antibodies. Using this procedure, the ubiquitinated forms of a given protein can be efficiently purified from mammalian cells, facilitating studies on the roles of ubiquitination in regulating protein function.

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.

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate ...

Measuring Ubiquitylated, SUMOylated, and ADP-Ribosylated DNA-Protein Crosslinks

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

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

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

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.

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

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

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

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.

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

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

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required 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-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

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

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.