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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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Ahmed, S., Holt, M., Riedel, D., Jahn, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-scale+isolation+of+synaptic+vesicles+from+mammalian+brain.">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 border="0" cellpadding="0" cellspacing="0" style="width:853px;" width="854">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
		</tr>
		<tr height="21">
			<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">
			<td height="21" style="height:21px;width:363px;">SUMOlink SUMO-1 Kit</td>
			<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">
			<td height="21" style="height:21px;width:363px;">QIAquick Gel Extraction Kit</td>
			<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>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Cell lifter</td>
			<td style="width:153px;">Corning</td>
			<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 ...

in-vitro-sumoylation-assay-to-study-sumo-e3-ligase-activity

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JoVE Journal

Biology

9.3K Views.  National Yang-Ming University. 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.

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.

Badania wizualne Wrażliwość na prędkość i kierunek ruchu w Jaszczurki

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

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...

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, ...

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the prediction of family functions based on several features, such as the presence of sequence motifs or of particular sites for post-translational modification and the expression profile of family members in different conditions. This work describes a detailed protocol for gene family characterization. Here, the procedure is applied to the characterization of the Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligase family in grapevine. The methods include the genome-wide identification of family members, the characterization of gene localization, structure, and duplication, the analysis of conserved protein motifs, the prediction of protein localization and phosphorylation sites as well as gene expression profiling across the family in different datasets. Such procedure, which could be extended to further analyses depending on experimental purposes, could be applied to any gene family in any plant species for which genomic data are available, and it provides valuable information to identify interesting candidates for functional studies, giving insights into the molecular mechanisms of plant adaptation to their environment.

This article describes the procedure for the identification and characterization of a gene family in grapevine applied to the family of Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligases.

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the ...