eoe sub articles

EoE Sub Articles

1. Preparation of Baculovirus Expression Constructs
<ol>
	<li>Purify SUMO E3 ligase K-bZIP by cloning the cDNA of K-bZIP 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 treatment.
	<ol>
		<li>Transfer the full-length K-bZIP cDNA into the dual expression vector with CpoI cloning sites. CpoI digests the PCR product and 1 &#181;g of the dual expression vector at 37 &#176;C for 3 h.</li>
		<li>Separate and extract the digested DNA following electrophoresis on a 0.8% agarose gel. Ligate the insert (K-bZIP) and vector by T4 DNA ligase at 16 &#176;C for 30 min according to the 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 h.</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 the 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 a 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, and 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 a 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;L.</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 the 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 a 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 staining. 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 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 film.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 E. coli DH5&#945;</td>
			<td>Yeastern Biotech</td>
			<td>FYE678-80VL</td>
			<td>competent E. coli cells A</td>
		</tr>
		<tr>
			<td>E. coli DH10Bac</td>
			<td>Invitrogen</td>
			<td>10359-016</td>
			<td>competent E. coli cells B</td>
		</tr>
		<tr>
			<td>FugeneHD</td>
			<td>Roche</td>
			<td>4709705001</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&#8217;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>Na2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>S5136-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</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>&#946;-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</td>
		</tr>
		<tr>
			<td>SUMOlink SUMO-2/3 Kit</td>
			<td>Active Motif</td>
			<td>40220</td>
			<td>standard SUMOylation protocol</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 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</td>
			<td>GE Healthcare</td>
			<td>28955810</td>
			<td></td>
		</tr>
		<tr>
			<td>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>

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.

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

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 ...

SUMOylation occurs due to the attachment of small ubiquitin-like modifiers, SUMOs, to substrate proteins.
SUMOylation occurs when a mature SUMO peptide is activated by an enzyme - E1. Upon activation, the SUMO is transferred to the active site of the conjugating enzyme - E2 - attaching the SUMO to the lysine residue of the substrate protein. Another ligase - E3 - enhances this interaction&#39;s efficiency.
To perform an in vitro SUMOylation assay, begin with a tube containing a mixture of the substrate protein - p53, enzymes, and SUMO peptides, and incubate to induce SUMOylation. Supplement the tube with sodium dodecyl sulfate - SDS-containing buffer, terminating the reaction. Heat the sample to denature the proteins.
Load the protein mix into the well of an SDS-PAGE gel and perform electrophoresis. Higher molecular weight SUMOylated proteins move slower than their non-SUMOylated variants, generating two distinct bands.
Transfer the gel onto a blotting membrane present on a wet filter paper in an electroblotting apparatus. Cover with a wet filter paper and electro-transfer the proteins from the gel onto the membrane.
Treat the membrane with anti-p53 antibodies, which bind to p53. Overlay with enzyme-conjugated secondary antibodies that bind to antibody-p53 complexes. Add a chemiluminescent substrate to generate luminescent product and image the membrane.
A higher molecular weight band corresponds to SUMOylated proteins, while the lower band corresponds to non-SUMOylated protein variants.

sumoylation-assay-an-vitro-technique-to-detect-sumoylation-status

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Immunoassays

163 Views. 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 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. 

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

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 ...

JoVE Journal

Biology

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

Transcript