Name: in vitro sumoylation assay to study sumo e3 ligase activity
Uploaded: 2018-01-29T12:30:00
Description: Watch this Scientific Journal Video about In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity at JoVE.com

This work was supported by grants from the Ministry of Science and Technology (MOST, 105-2320-B-010-007-MY3 to PCC), from the National Health Research Institute (NHRI-EX105-10215BC to PCC), from the Ministry of Science and Technology (MOST 105-2314-B-400-019 to HJK) and from the National Health Research Institute (NHRI MG-105-SP-10, NHRI MG-106-SP-10 to HJK). This work was also supported partly with National Yang-Ming University on manuscript publication to PCC. The funders had no role in study design, data collection and analysis, decision to publish, or reparation of the manuscript.

1. Preparation of Baculovirus Expression Constructs
<ol>
	<li>Purify SUMO E3 ligase K-bZIP by cloning the cDNA of K-bZIP19 into a dual expression baculovirus vector (see Table of Materials) with an N-terminal epitope-tag. We have had success using an octapeptide tag (indicated throughout the protocol as vector-tag-K-bZIP). 
	NOTE: The DNA template for K-bZIP polymerase chain reaction (PCR) cloning is cDNA reverse transcribed from RNA isolated from TREx F3H3-K-Rta BCBL-1 cel...

<ol>
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Y., 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=Role+of+SUMO-interacting+motif+in+Daxx+SUMO+modification,+subnuclear+localization,+and+repression+of+sumoylated+transcription+factors.">Role of SUMO-interacting motif in Daxx SUMO modification, subnuclear localization, and repression of sumoylated transcription factors.</a> Mol Cell. 24 (3), 341-354 (2006).</li><li>Welsh, M., Jamalpour, M., Zang, G., Akerblom, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+Src+Homology-2+domain+containing+protein+B+(SHB)+in+beta+cells.">The role of the Src Homology-2 domain containing protein B (SHB) in beta cells.</a> J Mol Endocrinol. 56 (1), 21-31 (2016).</li><li>Huang, W. Q., 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=Structure,+function,+and+pathogenesis+of+SHP2+in+developmental+disorders+and+tumorigenesis.">Structure, function, and pathogenesis of SHP2 in developmental disorders and tumorigenesis.</a> Curr Cancer Drug Targets. 14 (6), 567-588 (2014).</li><li>Perry, J. J., Tainer, J. A., Boddy, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+SIM-ultaneous+role+for+SUMO+and+ubiquitin.">A SIM-ultaneous role for SUMO and ubiquitin.</a> Trends Biochem Sci. 33 (5), 201-208 (2008).</li><li>Sun, H., Leverson, J. 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The in vitro SUMOylation protocol described here is routinely used to establish the SUMOylation status of identified Ubc9 substrates. The major limitation using the standard protocol to study the SUMOylation function of SUMO E3 ligase is the abundance of SUMO E1 activating and E2 conjugating enzymes Ubc9 that maximize the SUMO conjugation in in vitro systems. Considering this challenge, we believe that titration of the amount of SUMO E1 and E2 enzymes to the level that one can barely detect their activi...

SUMO modification was initially identified as a reversible post-translational modification (PTM) that regulates protein stability1. In addition to direct conjugation, SUMO can also be attached to a protein through non-covalent interaction by SUMO interaction motifs (SIMs)2. Similar to the binding of tyrosyl-phosphorylation by molecules harboring Src homology 2 (SH2) or phosphotyrosine binding (PTB) domains3,4, SUMO modification provides an additional interaction platform for selective recruitment of SIM-containing effector proteins5,6. In addition to regulation of signal transduction, SUMOylation of transcriptional factors and chromatin remodeling molecules serve to modulate gene expression7,8. Studies of genome-wide SUMOylation patterns have revealed that SUMO modification is associated with either positive9,10 or negative10,11,12 transcription regulation, in part due to the paralogue specificity of SUMOylation.
There are three major SUMO isoforms for protein conjugating present in mammalian cells; these include SUMO-1, and the highly homologous SUMO-2 and SUMO-3 (referred to as SUMO-2/3)13. SUMO is usually conjugated to the lysine (K) residue within the &#968;KxD/E consensus motif in the target protein. The SIM in SUMO E3 ligases is responsible for the paralogue specificity14. In contrast to ubiquitination pathways containing hundreds of E3 ligases, there have only been a few SUMO E3 ligases identified15. SUMO E3 ligases are defined by properties including their ability to (i) bind Ubc9, (ii) bind SUMO moiety via a SIM domain, and (iii) enhance SUMO transfer from Ubc9 onto substrate. E3 ligase is not absolutely required for SUMO conjugation16, but provides substrate and SUMO-paralogue specificity. Since usually only a small fraction of the total substrate protein is SUMO-modified, detection of SUMOylated proteins in vivo is always a challenge. Therefore, accurate and reproducible assays are needed in order to elucidate the precise function of SUMO modification.
The in vitro SUMOylation assay, which evaluates the ability of purified Ubc9 to catalyze SUMOylation of substrate proteins, is a well-accepted assay for studying SUMO modification17. However, SUMO E3 ligase activity in most cases cannot be detected or can only be detected with mutated SUMO ligase with high SUMO E3 ligase activity and low substrate specificity by the standard protocol because of the presence of an abundant amount of Ubc918. The overall success of this assay largely depends upon the careful titration of purified Ubc9. The in vitro SUMOylation assay described here is modified from a standard SUMOylation protocol (see Table of Materials). The observation of increased global SUMO modification during Kaposi&#39;s sarcoma-associated herpesvirus (KSHV) reactivation prompted us to identify the viral SUMO E3 ligase that may be responsible for the up-regulation of SUMOylation. Following a small-scale screening of the interacting proteins of viral SUMO E3 ligase K-bZIP, p53 was identified as a novel substrate. In this protocol, we show in detail in insect cells the steps involved in the expression and purification of wild-type K-bZIP, a viral SUMO E3 ligase with SUMO-2/3 specificity. The ability of the purified K-bZIP to enhance SUMOylation of p53, a well-known SUMO substrate, is evaluated in the presence of reduced amounts of E1 and E2 enzymes. Using this modified SUMOylation assay, researchers can reliably define the abilities of SUMO E3 ligase to SUMOylate novel or known substrates, which is a primary step in the study of protein SUMOylation. Moreover, this method helps identify novel SUMO E3 ligases with low ligase activity but high substrate specificity.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">pFastBac</td>
			<td style="width:153px;">Invitrogen</td>
			<td style="width:119px;">10359-016</td>
			<td style="width:219px;">dual expression baculovirus vector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCR2.1-TOPO vector</td>
			<td style="width:153px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td>PCR product vector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">competent cell&#160; E. coli DH5&#945;</td>
			<td style="width:153px;">Yeastern Biotech</td>
			<td style="width:119px;">FYE678-80VL</td>
			<td>competent E. coli cells A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">E. coli DH10Bac</td>
			<td style="width:153px;">Invitrogen</td>
			<td style="width:119px;">10359-016</td>
			<td>competent E. coli cells B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">FugeneHD</td>
			<td style="width:153px;">Roche</td>
			<td style="width:119px;">04709705001</td>
			<td>transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Opti-MEM</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">31985062</td>
			<td>reduced serum media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">T4 Ligase</td>
			<td style="width:153px;">NEW England BioLabs</td>
			<td style="width:119px;">M0202S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">CpoI (RsrII)</td>
			<td style="width:153px;">Thermo Scientific</td>
			<td style="width:119px;">ER0741</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Bluo-gal</td>
			<td style="width:153px;">Thermo Scientific</td>
			<td style="width:119px;">B1690</td>
			<td>galactosidase substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">IPTG</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">I6758-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Grace&#8217;s Insect Medium</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">11605094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Fetal Bovine Serum</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">10082147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Gentamicin</td>
			<td style="width:153px;">Thermo Fisher</td>
			<td style="width:119px;">15750060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Ampicillin</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9393-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Kanamycin</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">K0254-20ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">gentamicin</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">15710-064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">tetracyclin</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">87128-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">LB Broth</td>
			<td style="width:153px;">Merk</td>
			<td style="width:119px;">1.10285.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">HEPES</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">H4034-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">NaCl</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9888-5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">KCl</td>
			<td style="width:153px;">Merk</td>
			<td style="width:119px;">1.04936.1000</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:363px;">Na2HPO4</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5136-500G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:363px;">KH2PO4</td>
			<td style="width:153px;">J.T.Baker</td>
			<td style="width:119px;">3246-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">sodium dodecyl sulfate</td>
			<td style="width:153px;">Merk</td>
			<td style="width:119px;">1.13760.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">&#946;-mercaptoethanol</td>
			<td style="width:153px;">Bio-Rad</td>
			<td style="width:119px;">161-0710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">TRIS (Base)</td>
			<td style="width:153px;">J.T.Baker</td>
			<td style="width:119px;">4109-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Non-fat milk</td>
			<td style="width:153px;">Fonterra</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">glycerol</td>
			<td style="width:153px;">J.T.Baker</td>
			<td style="width:119px;">2136-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Triton X-100</td>
			<td style="width:153px;">Amresco</td>
			<td style="width:119px;">0694-1L</td>
			<td>detergent A</td>
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			<td height="21" style="height:21px;width:363px;">Tween 20</td>
			<td style="width:153px;">Amresco</td>
			<td style="width:119px;">0777-500ML</td>
			<td>detergent B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Poloxamer 188 solution</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5556-100ML</td>
			<td>detergent C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Protease Inhibitor Cocktail Tablet</td>
			<td style="width:153px;">Roche</td>
			<td style="width:119px;">04 693 132 001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">3x Flag peptide</td>
			<td style="width:153px;">Sigma</td>
			<td style="width:119px;">F4799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">anti-FLAG m2 Magnetic beads</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8823</td>
			<td>antibody-tagged magnetic beads</td>
		</tr>
		<tr height="21">
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			<td style="width:153px;">Active Motif</td>
			<td style="width:119px;">40120</td>
			<td>standard SUMOylation protocol&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">SUMOlink SUMO-2/3 Kit</td>
			<td style="width:153px;">Active Motif</td>
			<td style="width:119px;">40220</td>
			<td>standard SUMOylation protocol&#160;</td>
		</tr>
		<tr height="21">
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			<td style="width:153px;">QIAGEN</td>
			<td style="width:119px;">28704</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">QIAGEN Plasmid Mini Kit</td>
			<td style="width:153px;">QIAGEN</td>
			<td style="width:119px;">12123</td>
			<td>plasmid extraction kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Polypropylene tubes</td>
			<td style="width:153px;">Falcon</td>
			<td style="width:119px;">352059</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Petri Dish</td>
			<td style="width:153px;">Falcon</td>
			<td style="width:119px;">351029</td>
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		<tr height="21">
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			<td style="width:119px;">CNG3008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Loading tip</td>
			<td style="width:153px;">Sorenson BioScience</td>
			<td style="width:119px;">28480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">PVDF</td>
			<td style="width:153px;">PerkinElmer</td>
			<td style="width:119px;">NEF1002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Blotting filter paper</td>
			<td style="width:153px;">Bio-Rad</td>
			<td style="width:119px;">1703932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Mini slab gel apparatus (Bio-Rad Mini Protean II Cell)</td>
			<td style="width:153px;">Bio-Rad</td>
			<td style="width:119px;">1658001 EDU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell</td>
			<td style="width:153px;">Bio-Rad</td>
			<td style="width:119px;">1703940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Pierce ECL Western Blotting</td>
			<td style="width:153px;">Thermo</td>
			<td style="width:119px;">32106</td>
			<td>ECL reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:363px;">suspension mixer</td>
			<td style="width:153px;">Digisystem laboratory instruments&#160; Inc.</td>
			<td style="width:119px;">SM-3000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">orbital shaker</td>
			<td>Kansin instruments Co.</td>
			<td>OS701</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:363px;">ImageQuant LAS 4000 
			biomolecular imager</td>
			<td style="width:153px;">GE Healthcare</td>
			<td style="width:119px;">28955810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Sf9</td>
			<td style="width:153px;">Thermo Scientific</td>
			<td>B82501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">anti-p53 antibody</td>
			<td>Cell Signaling</td>
			<td>#9282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">anti-rabbit antibody</td>
			<td>GE Healthcare</td>
			<td>NA934-1ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro sumoylation assay to study sumo e3 ligase activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

According to the information provided by the manufacturer, the standard amount of E1 and E2 enzyme in the SUMOylation assay is 50 nM and 500 nM, respectively. The minimal amount of E2 conjugating enzyme Ubc9 that is able to SUMOylate p53 was first determined by an in vitro SUMOylation assay. As low as one-fifth of the amount of Ubc9 used in the standard in vitro SUMOylation assay protocol was able to efficiently SUMOylate p53 (Figure 1<stron...

Watch this Scientific Journal Video about In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity at JoVE.com

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

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

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...

The overall goal of this experiment is to verify a protein of interest that can be SUMOylated or is a SUMO E3 lygase. This method can help answer key questions in the SUMOylation field, such as SUMO E3 lygase identification. The main advantage of this technique is that it could be utilized for investigation of SUMO E3 lygase specificity toward different SUMO iso forms.Demonstrating the procedure will be Wan-Shan Young, a PhD student from my laboratory. To purify the tag K-bZIP, first incubate 10 milliliters of cellular lysate with 50 microliters of antibody-tagged magnetic beads in a 14-milliliter polypropylene tube. Then rotate the tube at 50 revolutions per minute at four degrees Celsius for three hours in a suspension mixer.Once the protein is captured centrifuge the sample at 800 times g for 30 seconds at four degrees Celsius to pellet the beads. Discard all but one milliliter of supernatant to re-suspend the beads. And transfer the dissolved beads into a 1.5-milliliter tube.To wash the captured proteins, place the tube with the beads on a magnetic stand. Wait for about three seconds for the beads to adhere to the side walls of the tube and then remove the supernatant. To wash the beads, add one milliliter of lysis buffer into the 1.5-milliliter tube.Then invert the tube ten times. Next, the place the 1.5-milliliter tube on the magnetic stand and remove the supernatant once the beads adhere to the walls. To wash the beads again, add one milliliter of phosphate buffered saline to the 1.5 milliliter tube, and invert it ten times.Then transfer the tube to the magnetic stand, and remove the supernatant once the beads have attached to the side walls. To elute the K-bZIP tagged proteins from the antibody-tagged magnetic beads, add 100 microliters at 150 micrograms per milliliter of octapeptide diluted in phosphate buffered saline in the 1.5-milliliter tube. Then rotate the tube on a suspension mixer at 50 revolutions per minute for ten minutes at room temperature.Transfer the tube to the magnetic stand. And then collect the K-bZIP containing supernatant. Next, to analyze the purified K-bZIP tag, subject one to five microliters of the purified protein to SDS page, followed by Coomassie blue staining.Load 0.5, 1 and 2 micrograms of bovine serum albumin as standards on the gel for quantifying the K-bZIP concentration. While processing the image on Image J.Next, dilute the purified K-bZIP in phosphate buffered saline. To attain a final concentration of 100 nanograms per microliter.And store at minus 80 degrees Celsius in small aliquots. Add three microliters of 100 nanograms per microliter of purified tagged K-bZIP in the in vitro SUMOylation master mix reaction. Then mix the contents of the master mix gently and incubate at 30 degrees Celsius for three hours.Next, add 20 microliters of 2X SDS page with loading buffer to stop the reaction. Then heat the samples at 95 degrees Celsius for five minutes to denature the protein. Next, load 20 microliters of the sample on a 10%SDS page gel.And run the gel at 80 volts for about 120 minutes till the dye reaches the bottom of the gel. Once the electro paresis is over, disconnect the gel apparatus. And transfer the gel in semi-dry transfer buffer for five minutes.Then soak the PVDF membrane in another container filled with methanol for one minute. Remove the PVDF membrane from the methanol and soak in semi-dry transfer buffer. Then gently agitate the membrane for five minutes.Remove the safety cover of the semi-dry electrophoretic apparatus. Pre-wet a filter paper. And prepare a gel sandwich on the bottom of the platinum anode.After securing the cathode plate in the safety cover, run the blot at 15 volts constant current for 90 minutes. Then turn the power supply off and take the PVDF membrane out after disconnecting the semi-dry apparatus. Now block the PVDF membrane with blocking buffer for one hour at room temperature on an orbital shaker at 30 revolutions per minute.Next, hybridize the PVDF membrane with anti-p53 antibody and blocking buffer for 12 to 16 hours at four degrees Celsius on a suspension mixer at 30 revolutions per minute. Then take the PVDF membrane out and transfer in a container filled with Tris-buffered saline tween 20. Next, soak the PVDF membrane in Tris-buffered saline tween 20 for 30 minutes on the orbital shaker at 45 revolutions per minute.Then hybridize the PVDF membrane with anti-rabbit antibody conjugated with horseradish peroxidase diluted in blocking buffer for one hour at room temperature on the suspension mixer at 30 revolutions per minute. Next, wash the PVDF membrane with Tris-buffered saline tween 20 thrice. After washing, soak the PVDF membrane in Tris-buffered saline tween 20 for 30 minutes on the suspension mixer at 45 revolutions per minute.Then replace the Tris-buffered saline tween 20 with phosphate-buffered saline. To preserve the PVDF membrane at four degrees Celsius for up to 12 hours. Next, mix the enhanced chemiluminescent substrate reagents one and two in a one-to-one ratio.Then remove the PVDF membrane from the phosphate-buffered saline and blot briefly with the punch pockets to absorb the excess moisture. Immediately add 400 microliters of the Chemiluminescent reagent to the membrane surface and wait for three to five minutes. Briefly blot to drain the excess Chemiluminescent reagent, preventing the membrane from drying completely.Use Luminescent's imaging system to expose the blot. Here immuno blot analysis was done to show the concentration of Ubc9 enzyme required to SUMOylate the p53 targets. This shows that 1/2 and 1/5 the concentration of Ubc9 enzyme can sufficiently SUMOylate p53 enzyme in an in vitro SUMOylation assay using SUMO 1 and SUMO 2 peptides.To analyze the SUMOylated p53 the membrane was probed with anti-p53 antibody. Similar immuno blot analysis was done to show that half of E1 and 1/10 concentration of Ubc9 enzymes are required to SUMOylate the p53 target in an in vitro SUMOylation assay using SUMO 1, SUMO 2 and SUMO 3 peptides. Here the membrane was stained with Coomassie to show the purified K-bZIP protein.Baculovirus expression system was used to express the purified K-bZIP which was analyzed on SDS page with bovine serum albumin as standard for comparison. Additional immuno blot analysis was also done which showed that on increasing the concentration of K-bZIP it catalyzes the SUMOylation of p53 in the presence of half the amount of E1, and 1/10 the amount of Ubc9 enzymes, respectively. Once mastered, this technique can be done in four hours if it is performed properly.While attempting this procedure it is important to remember to keep all the reagent for the in vitro SUMOylation assay on ice, and to do the series dilution rather than in one giant dilution. Find this procedure other methods like over expression SUMOlygase can be performed in order to verify the SUMOylation in vitro. After its development, this technique paved the way for researchers in the field of SUMOylation to explore more SUMOlygase and validate their power of specificity.After watching this video, you should have a good understanding of how to identify a SUMO e3 lygase using in vitro simulation assay.

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