This work was supported by NIH grants RO1 DC006497, RO1 NS057289, and PO1 ES016738 (to Z. Zhang); California Institute for Regenerative Medicine grants RS1-00331-1 and RL1-00682-1 (to Z. Zhang); the American Parkinson Disease Association (to Z. Zhang and Y. S. Choo); and the Michael J. Fox Foundation for Parkinson&rsquo;s Research (Z. Zhang).

Detection of protein ubiquitination in cultured cells
<ol>
<li>Transfect cultured cells with plasmids expressing the protein of interest and (epitope-tagged version of) ubiquitin.</li>
<li>Make complete cell lysis buffer (2% SDS, 150 mM NaCl, 10 mM Tris-HCl, pH 8.0) with 2mM sodium orthovanadate, 50 mM sodium fluoride, and protease inhibitors.</li>
<li>Lyse cells with 100 &micro;l cell lysis buffer per plate (6 cm dish). If a larger dish is used, adjust volume accordingly.&nbsp; Swirl the dish carefully to let the lysis buffer cover the entire area of grown cells.</li>
<li>Collect the cells with a cell scraper and transfer the cell lysates into a 1.5 ml eppendorf tube. Place the tube onto a hot plate immediately to boil for 10 min.</li>
<li>Shear the cells with a sonication device.</li>
<li>Add 900ul of dilution buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton). Incubate samples at 4&deg;C for 30-60 min with rotation.</li>
<li>Spin the diluted samples at 20,000 x g for 30 min. Transfer the resulting supernatant to a new eppendorf tube. Be careful not to disturb the pellet.</li>
<li>Measure the protein concentration.</li>
<li>Prepare Protein A- or G-agarose bead-conjugated antibody against the target protein in a compatible buffer (50% slurry). Cut the narrow end of a P-200 pipette tip and transfer 14-20 &mu;l of resin to 500-1,500 &mu;g of prepared cell lysates for immunoprecipitation. For cells with high transfection efficiency, 500 &mu;g will be enough. For cells with low transfection efficiency, more protein may be needed.</li>
<li>Incubate the cell lysate-bead mixture at 4&deg;C overnight with rotation.</li>
<li>Spin down the beads at 5000 x g for 5 min. Aspirate the supernatant. Wash the resin with the washing buffer (10 mM Tris-HCl, pH 8.0, 1 M NaCl, 1 mM EDTA, 1% NP-40) twice.</li>
<li>Spin the beads for a final time at 20,000 x g for 30 sec. Aspirate the residual washing buffer and boil the resin with 2X SDS loading buffer.</li>
<li>Load samples onto a SDS-PAGE gel for immunoblotting analysis.</li>
<li>Detect ubiquitin and the target protein with respective antibodies. For immunoblotting, we generally detect ubiquitin first. The membrane will then be used to detect the protein precipitated.</li>
</ol>
Detection of ubiquitination on target protein through In vitro ubiquitination assay
<ol>
<li>Make 5X ubiquitination buffer (100 mM Tris-HCl, pH 7.5, 25 mM MgCl2, 2.5 mM DTT, 10 mM ATP). Store them in small aliquots at -20 &deg;C for up to 6 month. Some protocols also add creatine phosphate and creatine kinase into the buffer for ATP regeneration 1,2.</li>
<li>For each reaction, prepare a mixture containing the following: 
<table border="1">
<tbody>
<tr>
<td>8 &mu;l</td>
<td>5X ubiquitination buffer</td>
</tr>
<tr>
<td>250 ng</td>
<td>ubiquitination E1</td>
</tr>
<tr>
<td>500 ng</td>
<td>ubiquitination E2</td>
</tr>
<tr>
<td>0.5 &mu;g</td>
<td>ubiquitin</td>
</tr>
<tr>
<td>0.5 &mu;g</td>
<td>protein of interest</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>water to 40 &mu;l total volume</td>
</tr>
</tbody>
</table>
As controls, prepare similar reactions in the absence of either E1, E2, or ubiquitin.</li>
<li>Incubate the mixture at 37&deg;C for 1 hour or longer.</li>
<li>Stop the reaction by adding SDS-PAGE sample buffer and boil the sample for 10 min.</li>
<li>Load samples onto SDS-PAGE gel for immunoblotting analysis.</li>
<li>Detect ubiquitin and the target protein with respective antibodies.</li>
</ol>

<ol>
	<li>Xiong, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINK1,+and+DJ-1+form+a+ubiquitin+E3+ligase+complex+promoting+unfolded+protein+degradation.">PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation.</a> J Clin Invest. 119, 650-660 (2009).</li><li>Xirodimas, D. P., Saville, M. K., Bourdon, J. C., Hay, R. T., Lane, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mdm2-mediated+NEDD8+conjugation+of+p53+inhibits+its+transcriptional+activity.">Mdm2-mediated NEDD8 conjugation of p53 inhibits its transcriptional activity.</a> Cell. 118, 83-97 (2004).</li><li>Didier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNF5,+a+RING+finger+protein+that+regulates+cell+motility+by+targeting+paxillin+ubiquitination+and+altered+localization.">RNF5, a RING finger protein that regulates cell motility by targeting paxillin ubiquitination and altered localization.</a> Mol Cell Biol. 23, 5331-5345 (2003).</li><li>Laney, J. D., Hochsetrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unit+14.5+Analysis+of+Protein+Ubiquitination.">Unit 14.5 Analysis of Protein Ubiquitination.</a> Current Protocols in Protein Science. , 14.5.1-14.5.11 (2002).</li></ol>

The authors have nothing to disclose.

In this presentation, we first described steps that permit detecting ubiquitin modification on a protein of interest in cultured mammalian cells. In order to detect ubiquitination specifically on the protein of interest, not on non-covalently interacting proteins, we used a stringent condition for cell lysis, immunoprecipitation and washing 1,3.&nbsp; Ubiquitination, detected by immunoprecipitation of target protein in such a harsh condition followed by anti-ubiquitin immunoblotting, is therefore likely to be ...

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.

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Detection of Protein Ubiquitination

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 ...

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42.6K Views.  The Sanford Burnham Institute for Medical Research. 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.