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>

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.

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154 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. 

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

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