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 cells following doxycycline treatment20.
	<ol>
		<li>Transfer the full-length K-bZIP cDNA19 into the dual expression vector with CpoI cloning sites. CpoI digest the PCR product and 1 &#181;g of the dual expression vector at 37 &#176;C for 3 h21.</li>
		<li>Separate and extract the digested DNA following electrophoresis on a 0.8% agarose gel22. Ligate the insert (K-bZIP) and vector by T4 DNA ligase at 16 &#176;C for 30 min according to manufacturer&#39;s instructions (see Table of Materials).</li>
		<li>Add 5 &#181;L of ligation DNA to 50 &#181;L competent E. coli cells &#34;A&#34; (see Table of Materials) in a 1.5 mL tube. Put on ice for 30 min. Then, incubate the 1.5 mL tube in a 42 &#176;C water bath for 45 s and immediately put the tube on ice for 3 min.</li>
		<li>Add 600 &#181;L S.O.C. medium (0.5% (w/v) yeast extract, 2% (w/v) tryptone, 10 mM NaCl, 2.5 mM KCl, 20 mM MgSO4, and 4% (w/v) glucose) into the tube and incubate at 37 &#176;C for 1 h. Then, centrifuge the tube at room temperature for 3 min at 600 x g.</li>
		<li>Remove supernatant, resuspend pellet with 60 &#181;L Luria-Bertani medium (LB; 1% (w/v) tryptone, 0.5% (w/v) yeast extract, and 85 mM NaCl) and spread on LB agar plates containing 100 &#181;g/mL ampicillin. Incubate the agar plate at 37 &#176;C for 16 h23,24.</li>
		<li>Select 1 colony to inoculate in 5 mL LB medium containing 100 &#181;g/mL ampicillin and culture at 37 &#176;C for 16 h with 250 rpm shaking. Then, extract the plasmid, vector-tag-K-bZIP, by a plasmid extraction kit according to the manufacturer&#39;s instructions (see Table of Materials).</li>
	</ol>
	</li>
	<li>Generate the recombinant bacmid harboring the tag-K-bZIP by transformation of the vector-tag-K-bZIP into competent E. coli cells &#34;B&#34; (see Table of Materials).
	<ol>
		<li>Mix 0.2 &#181;g vector-tag-K-bZIP plasmid and 100 &#181;L competent E. coli cells &#34;B&#34; in a 1.5 mL tube and put on ice for 30 min. Incubate the tube in 42 &#176;C water bath for 45 s and immediately put the tube on ice for 3 min.</li>
		<li>Add 900 &#181;L of S.O.C. medium into the tube and incubate at 37 &#176;C for 4 h with rotation at 50 rpm. Next, take 10 &#181;L of the medium, add an additional 50 &#181;L of S.O.C medium to spread on LB agar plates containing 50 &#181;g/mL kanamycin, 7 &#181;g/mL gentamicin, 10 &#181;g/mL tetracycline, 100 &#181;g/mL galactosidase substrate, and 40 &#181;g/mL isopropyl &#946;-D-1-thiogalactopyranoside (IPTG), and incubate the plates for 48 h at 37 &#176;C.</li>
		<li>Inoculate one white colony in 5 mL LB medium containing 50 &#181;g/mL kanamycin, 7 &#181;g/mL gentamicin, and 10 &#181;g/mL tetracycline. Incubate at 37 &#176;C with 250 rpm shaking for 16 h.</li>
		<li>Isolate the recombinant bacmid DNA harboring the tag-K-bZIP, quantify, and re-suspend at a concentration of 1 &#181;g/&#181;L25.</li>
	</ol>
	</li>
	<li>Generate recombinant baculoviruses expressing tag-K-bZIP by transfecting 1 &#181;g of recombinant bacmid DNA containing tag-K-bZIP into Sf9 cells in one well of a 6-well plate.
	<ol>
		<li>Seed 2 x 106 Sf9 cells (see Table of Materials) in 2 mL Grace&#39;s Insect Medium supplied with 10% fetal bovine serum (FBS) and 1% gentamicin in each well of a 6 well plate, then incubate at 27 &#176;C for 1 h.</li>
		<li>Maintain a log phase culture of Sf9 cells in Grace&#39;s Insect Medium supplied with 10% FBS, 1% gentamicin, and 1% detergent C (see Table of Materials) at 27 &#176;C in orbital suspension at 140 rpm. Count cells using a hemocytometer and trypan blue staining; cell viability should exceed 95%. Maintain cells using a subcultivation ratio of 1:3 every 2-3 days (minimal density ~0.5 x 106 cells/mL).</li>
		<li>Add 2 &#181;L bacmid DNA (1 &#181;g/&#181;L) and 98 &#181;L reduced serum media (see Table of Materials) into a 2 mL tube. Then, add 4 &#181;L transfection reagent (see Table of Materials) to the tube and mix well. Incubate at room temperature for 15 min.</li>
		<li>Add this transfection mixture (104 &#181;L) into each well, and incubate at 27 &#176;C for 12-16 h.</li>
		<li>Remove exhausted media with gentle aspiration, and replace with 2 mL of fresh Grace&#39;s Insect Medium supplied with 10% FBS and 1% gentamicin. The Sf9 cells adhere loosely to the plate surface and will not be disturbed with gentle aspiration.</li>
	</ol>
	</li>
	<li>Collect recombinant baculovirus after transfection and amplify baculovirus to obtain high-titer stocks (P1) for further experiments.
	<ol>
		<li>72 h after changing medium, scrape the Sf9 cells using a sterile cell lifter, collect the supernatant from each individual well in a 1.5 mL tube and vortex for 10 s.</li>
		<li>Centrifuge at 4 &#176;C for 3 min at 150 x g. Collect supernatant as P0 baculovirus and aliquot in 1.5 mL tubes (1 mL baculovirus in each tube). Store at -80 &#176;C.</li>
		<li>Seed 1 x 107 Sf9 cells in 10 mL Grace&#39;s Insect Medium supplied with 10% FBS and 1% gentamicin in a 10 cm Petri dish and incubate at 27 &#176;C for 1 h.</li>
		<li>For amplification of recombinant baculoviruses expressing tag-K-bZIP, add 0.5 mL P0 baculovirus to the seeded Sf9 cells and incubate at 27 &#176;C for 48 h.</li>
		<li>Collect supernatant as P1 baculovirus in a 15 mL tube and store at 4 &#176;C for up to 1 month.</li>
	</ol>
	</li>
</ol>
2. Purification of K-bZIP
<ol>
	<li>Maintain a log phase culture of Sf9 cells in Grace&#39;s Insect Medium supplied with 10% FBS, 1% gentamicin, and 1% detergent C at 27 &#176;C in orbital suspension at 140 rpm as described in step 1.3.2.</li>
	<li>On the day of transduction, add 1 mL of baculovirus-containing supernatant (P1) into 250 mL of Sf9 cells with a density of 2 x 106 cells/mL.</li>
	<li>Incubate Sf9 cells for 72 h at 27 &#176;C on an orbital shaker with 140 rpm shaking, then collect by centrifugation at 2,740 x g for 15 min at 4 &#176;C.</li>
	<li>Remove supernatant and lyse cell pellets in 10 mL lysis buffer (20 mM HEPES pH 7.9, 0.5 M NaCl, 1% detergent A (see Table of Materials), 2% glycerol, protease inhibitor cocktail) and rotate at 50 rpm using a suspension mixer at 4 &#176;C for 30 min.</li>
	<li>Centrifuge cell lysate at 15,000 x g for 15 min to collect the supernatant.</li>
	<li>To purify tag-K-bZIP, incubate the cell lysates (10 mL) with 50 &#181;L of antibody-tagged magnetic beads in 14 mL polypropylene tubes, and rotate at 50 rpm for 3 h at 4 &#176;C using a suspension mixer. Following protein capture, pellet the beads at 800 x g centrifugation for 30 s at 4 &#176;C. Remove most of the supernatant, re-suspend the beads in the residual volume of about 1 mL, and transfer to a 1.5 mL tube for washing.</li>
	<li>Wash the captured protein by placing the tube containing the beads on a magnetic stand, and remove the supernatant once the magnetic beads have fully adhered to the side of the tube.</li>
	<li>Wash the beads with lysis buffer four times.
	<ol>
		<li>Add 1 mL lysis buffer into 1.5 mL tubes, and invert the tubes 10 times. Place the 1.5 mL tube on a magnetic stand and remove the supernatant as described in 2.7.</li>
		<li>Repeat the previous step another 3 times.</li>
	</ol>
	</li>
	<li>Wash the beads with phosphate buffered saline (PBS) once.
	<ol>
		<li>Add 1 mL PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4) into 1.5 mL tubes, and invert the tubes 10 times.</li>
		<li>Put the 1.5 mL tube on a magnetic stand and then remove the supernatant.</li>
	</ol>
	</li>
	<li>Elute tag-K-bZIP protein from the antibody-tagged magnetic beads by adding 100 &#181;L of 150 &#181;g/mL octapeptide diluted in PBS into a 1.5 mL tube. Rotate the tube for 10 min at 50 rpm by suspension mixer at room temperature. Then, place the tube back on the magnetic stand and collect the K-bZIP-containing supernatant in a clean 1.5 mL tube.</li>
	<li>Analyze 1-5 &#181;L purified tag-K-bZIP protein by SDS-PAGE followed by Coomassie blue staining26. Load 2 &#181;g, 1 &#181;g, and 0.5 &#181;g samples of bovine serum albumin (BSA) on the same gel and use it for quantification with an image processing program, like ImageJ. Estimate the K-bZIP concentration by comparison to the BSA standards.</li>
	<li>The purified K-bZIP is now ready to be used in each SUMOylation assay. Dilute the K-bZIP in PBS to a final concentration of 100 ng/&#181;L and store in small aliquots at -80 &#176;C.</li>
</ol>
3. SUMOylation Assay
<ol>
	<li>Add 3 &#181;L of 100 ng/&#181;L of purified tag-K-bZIP into the master mix of each in vitro SUMOylation reaction. Components in master mix contain 1 &#181;L protein buffer, 1x SUMOylation buffer, 0.5 &#181;L p53 protein (0.5 mg/mL), 25 nM of E1 activating enzyme (stock conc.: 1 &#181;M), 50 nM of E2 conjugating enzyme (stock conc.: 10 &#181;M), and 250 nM each of the SUMO peptides (SUMO-1, SUMO-2, or SUMO-3; stock conc.: 5 &#181;M) to a final volume of 17 &#181;L.</li>
	<li>Mix the contents gently and incubate the reaction at 30 &#176;C for 3 h. Stop the reaction by adding 20 &#181;L of 2x SDS-PAGE loading buffer (100 mM Tris-HCL pH 6.8, 4% sodium dodecyl sulfate, 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol, and 200 mM &#946;-mercaptoethanol) and denature the samples at 95 &#176;C for 5 min.</li>
	<li>Separate samples on SDS-PAGE, transfer the separated proteins onto PVDF membrane by using a semi-dry electrophoretic transfer apparatus, and analyze by Western blot using anti-p53 antibody.
	<ol>
		<li>Set up the gel apparatus as in step 2.11, and load 20 &#181;L of sample into gel wells.</li>
		<li>Run the 10% gel in a constant current at 80 V for about 120 min (until the dye band reaches the bottom of the gel). After completion of electrophoresis, turn off the power supply.</li>
		<li>Disconnect the gel apparatus and gel cassettes to take out the gel, and float the gel into semi-dry transfer buffer (48 mM Tris-HCl, 39 mM glycine, 20% methanol) for 5 min.</li>
		<li>Take another container and soak the PVDF membrane in methanol for 1 min. Then, take PVDF out of methanol and put in semi-dry transfer buffer. Gently agitate the membrane for 5 min.</li>
		<li>Remove the safety cover of the semi-dry electrophoretic apparatus.</li>
		<li>Pre-wet filter paper, and prepare a gel sandwich on the bottom platinum anode as follows: filter paper, PVDF membrane, gel, and filter paper.</li>
		<li>Secure cathode plate and safety cover, then run blot at 15 V constant current for 90 min. Turn off the power supply, disconnect semi-dry apparatus, and take out the PVDF membrane.</li>
		<li>Block PVDF membrane with blocking buffer (5% non-fat milk in TBST buffer (137 mM NaCl2, 20 mM Tris-HCl, 0.1% detergent B (see Table of Materials), pH 7.6)) at room temperature for 1 h with 30 rpm shaking by orbital shaker.</li>
		<li>Hybridize the PVDF membrane with anti-p53 antibody diluted (1:1,000) in blocking buffer for 12-16 h at 4 &#176;C with 30 rpm shaking using a suspension mixer.</li>
		<li>Take out the PVDF membrane into a container and put in TBST. Rinse PVDF membrane with TBST twice more.</li>
		<li>Soak the PVDF membrane in TBST for 30 min with 45 rpm shaking by orbital shaker.</li>
		<li>Hybridize the PVDF membrane with anti-rabbit antibody conjugated with horseradish peroxidase (HRP) diluted (1:4,000) in blocking buffer for 1 h at room temperature with 30 rpm shaking using a suspension mixer.</li>
		<li>Wash the PVDF membrane with TBST 3 times as described in step 3.4.10.</li>
		<li>Soak the PVDF membrane in TBST for 30 min with 45 rpm shaking using a suspension mixer. Remove TBST and add PBS to preserve the PVDF membrane at 4 &#176;C for up to 12 h.</li>
		<li>Mix the enhanced chemiluminescent substrate (ECL substrate) reagent 1 and 2 (1:1) (see Table of Materials). Remove the PVDF membrane from the PBS, blot briefly with a paper towel or light-duty wiper to absorb excess moisture, and immediately add 400 &#181;L of ECL reagent to the surface of each membrane for 3-5 min. Remove excess ECL reagent by briefly blotting, but do not allow the membrane to completely dry.</li>
		<li>Expose the blot using a luminescence imaging system or autoradiography film27.</li>
	</ol>
	</li>
</ol>

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<li>Ahmed, S., Holt, M., Riedel, D., Jahn, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small-scale+isolation+of+synaptic+vesicles+from+mammalian+brain%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Small-scale isolation of synaptic vesicles from mammalian brain.</a> Nat Protoc. 8 (5), 998-1009 (2013).</li>
</ol>

The authors disclose no potential conflicts of interest.

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.

<table><tbody><tr><td>pFastBac</td><td>Invitrogen</td><td>10359-016</td><td>dual expression baculovirus vector</td></tr><tr><td>pCR2.1-TOPO vector</td><td>Invitrogen</td><td></td><td>PCR product vector</td></tr><tr><td>competent cell&amp;nbsp; E. coli DH5&amp;alpha;</td><td>Yeastern Biotech</td><td>FYE678-80VL</td><td>competent &lt;em&gt;E. coli&lt;/em&gt; cells A</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; DH10Bac</td><td>Invitrogen</td><td>10359-016</td><td>competent &lt;em&gt;E. coli&lt;/em&gt; cells B</td></tr><tr><td>FugeneHD</td><td>Roche</td><td>04709705001</td><td>transfection reagent</td></tr><tr><td>Opti-MEM</td><td>Gibco</td><td>31985062</td><td>reduced serum media</td></tr><tr><td>T4 Ligase</td><td>NEW England BioLabs</td><td>M0202S</td><td></td></tr><tr><td>CpoI (RsrII)</td><td>Thermo Scientific</td><td>ER0741</td><td></td></tr><tr><td>Bluo-gal</td><td>Thermo Scientific</td><td>B1690</td><td>galactosidase substrate</td></tr><tr><td>IPTG</td><td>Sigma-Aldrich</td><td>I6758-1G</td><td></td></tr><tr><td>Grace&amp;rsquo;s Insect Medium</td><td>Gibco</td><td>11605094</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>10082147</td><td></td></tr><tr><td>Gentamicin</td><td>Thermo Fisher</td><td>15750060</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>A9393-25G</td><td></td></tr><tr><td>Kanamycin</td><td>Sigma-Aldrich</td><td>K0254-20ML</td><td></td></tr><tr><td>gentamicin</td><td>Gibco</td><td>15710-064</td><td></td></tr><tr><td>tetracyclin</td><td>Sigma-Aldrich</td><td>87128-25G</td><td></td></tr><tr><td>LB Broth</td><td>Merk</td><td>1.10285.0500</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H4034-100G</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9888-5KG</td><td></td></tr><tr><td>KCl</td><td>Merk</td><td>1.04936.1000</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S5136-500G</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>J.T.Baker</td><td>3246-01</td><td></td></tr><tr><td>sodium dodecyl sulfate</td><td>Merk</td><td>1.13760.1000</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Bio-Rad</td><td>161-0710</td><td></td></tr><tr><td>TRIS (Base)</td><td>J.T.Baker</td><td>4109-06</td><td></td></tr><tr><td>Non-fat milk</td><td>Fonterra</td><td></td><td></td></tr><tr><td>glycerol</td><td>J.T.Baker</td><td>2136-01</td><td></td></tr><tr><td>Triton X-100</td><td>Amresco</td><td>0694-1L</td><td>detergent A</td></tr><tr><td>Tween 20</td><td>Amresco</td><td>0777-500ML</td><td>detergent B</td></tr><tr><td>Poloxamer 188 solution</td><td>Sigma-Aldrich</td><td>P5556-100ML</td><td>detergent C</td></tr><tr><td>Protease Inhibitor Cocktail Tablet</td><td>Roche</td><td>04 693 132 001</td><td></td></tr><tr><td>3x Flag peptide</td><td>Sigma</td><td>F4799</td><td></td></tr><tr><td>anti-FLAG m2 Magnetic beads</td><td>Sigma-Aldrich</td><td>M8823</td><td>antibody-tagged magnetic beads</td></tr><tr><td>SUMOlink SUMO-1 Kit</td><td>Active Motif</td><td>40120</td><td>standard SUMOylation protocol&amp;nbsp;</td></tr><tr><td>SUMOlink SUMO-2/3 Kit</td><td>Active Motif</td><td>40220</td><td>standard SUMOylation protocol&amp;nbsp;</td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>QIAGEN</td><td>28704</td><td></td></tr><tr><td>QIAGEN Plasmid Mini Kit</td><td>QIAGEN</td><td>12123</td><td>plasmid extraction kit</td></tr><tr><td>Polypropylene tubes</td><td>Falcon</td><td>352059</td><td></td></tr><tr><td>Petri Dish</td><td>Falcon</td><td>351029</td><td></td></tr><tr><td>Cell lifter</td><td>Corning</td><td>CNG3008</td><td></td></tr><tr><td>Loading tip</td><td>Sorenson BioScience</td><td>28480</td><td></td></tr><tr><td>PVDF</td><td>PerkinElmer</td><td>NEF1002</td><td></td></tr><tr><td>Blotting filter paper</td><td>Bio-Rad</td><td>1703932</td><td></td></tr><tr><td>Mini slab gel apparatus (Bio-Rad Mini Protean II Cell)</td><td>Bio-Rad</td><td>1658001 EDU</td><td></td></tr><tr><td>Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell</td><td>Bio-Rad</td><td>1703940</td><td></td></tr><tr><td>Pierce ECL Western Blotting</td><td>Thermo</td><td>32106</td><td>ECL reagent</td></tr><tr><td>suspension mixer</td><td>Digisystem laboratory instruments&amp;nbsp; Inc.</td><td>SM-3000</td><td></td></tr><tr><td>orbital shaker</td><td>Kansin instruments Co.</td><td>OS701</td><td></td></tr><tr><td>ImageQuant LAS 4000&lt;br/&gt; biomolecular imager</td><td>GE Healthcare</td><td>28955810</td><td></td></tr><tr><td>Sf9</td><td>Thermo Scientific</td><td>B82501</td><td></td></tr><tr><td>anti-p53 antibody</td><td>Cell Signaling</td><td>#9282</td><td></td></tr><tr><td>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 ...

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Biology

9.3K Views.  National Yang-Ming University. In tegenstelling tot ubiquitin ligases, zijn paar E3 SUMO ligases geïdentificeerd. Deze gewijzigde in vitro SUMOylation protocol vermag roman SUMO E3 ligases identificeren door een reconstructie in vitro assay.

Video: In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

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. ...

Biochemistry

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in liver cancer, the modification by SUMO (Small Ubiquitin MOdifier) has been given the most attention. Isolation of endogenous SUMOylated proteins in vivo is challenging due to the presence of active SUMO-specific proteases. Initial studies of SUMOylation in vivo were based on the molecular detection of specific SUMOylated proteins (e.g., by western blot). However, in many cases, antibodies, generally made with non-modified recombinant protein, did not immunoprecipitate SUMOylated forms of the protein of interest.&#160;Nickel chromatography has been the other approach to study SUMOylation by capturing histidine-tagged versions of SUMO molecules. This approach is mainly used in cells stably expressing or transiently transfected with His-SUMO molecules. To overcome these limitations, SUMO-binding entities (SUBEs) were developed to isolate endogenous SUMOylated proteins. Herein, we describe all the steps required for the enrichment, isolation, and identification of SUMOylated substrates from human hepatoma cells and hepatic tissues from a liver cancer mouse model by using SUBEs. Firstly, we describe methods involved in the preparation and lysis of the human hepatoma cells and liver tumor tissue samples. Then, a thorough explanation of the preparation of SUBEs and controls is detailed along with the protocol for the protein pull-down assays. Finally, some examples are provided regarding the options available for the identification and characterization of the SUMOylated proteome, namely the use of western-blot analysis for the detection of a specific SUMOylated substrate from liver tumors or the use of proteomics by mass spectrometry for high-throughput characterization of the SUMOylated proteome and interactome in hepatoma cells.

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Here, we present a protocol to enrich, isolate, identify, and characterize proteins modified by SUMO in vivo both from human hepatoma cells and liver tumors obtained from mouse models of hepatocellular carcinoma by using SUMO-binding entities (SUBEs).

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...

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, ...

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 ...

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.

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 ...

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

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

Source: Yang, W. S. et al., <a href="https://app.jove.com/v/56629">In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity.</a> J. Vis. Exp. (2018)
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.

Source: Yang, W. S. et al., In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity. J. Vis. Exp. (2018)
This video demonstrates the in vitro method ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

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