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

1. Preparation of Baculovirus Expression Constructs

1. 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.
   1. Transfer the full-length K-bZIP cDNA into the dual expression vector with CpoI cloning sites. CpoI digests the PCR product and 1 µg of the dual expression vector at 37 °C for 3 h.
   2. 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 °C for 30 min according to the manufacturer's instructions (see Table of Materials).
   3. Add 5 µL of ligation DNA to 50 µL competent E. coli cells "A" (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 °C water bath for 45 s and immediately put the tube on ice for 3 min.
   4. Add 600 µL 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 °C for 1 h. Then, centrifuge the tube at room temperature for 3 min at 600 x g.
   5. Remove supernatant, resuspend pellet with 60 µL 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 µg/mL ampicillin. Incubate the agar plate at 37 °C for 16 h.
   6. Select 1 colony to inoculate in 5 mL LB medium containing 100 µg/mL ampicillin and culture at 37 °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's instructions (see Table of Materials).
2. Generate the recombinant bacmid harboring the tag-K-bZIP by the transformation of the vector-tag-K-bZIP into competent E. coli cells "B" (see Table of Materials).
   1. Mix 0.2 µg vector-tag-K-bZIP plasmid and 100 µL competent E. coli cells "B" in a 1.5 mL tube and put on ice for 30 min. Incubate the tube in a 42 °C water bath for 45 s and immediately put the tube on ice for 3 min.
   2. Add 900 µL of S.O.C. medium into the tube and incubate at 37 °C for 4 h with rotation at 50 rpm. Next, take 10 µL of the medium, and add an additional 50 µL of S.O.C medium to spread on LB agar plates containing 50 µg/mL kanamycin, 7 µg/mL gentamicin, 10 µg/mL tetracycline, 100 µg/mL galactosidase substrate, and 40 µg/mL isopropyl β-D-1-thiogalactopyranoside (IPTG), and incubate the plates for 48 h at 37 °C.
   3. Inoculate one white colony in a 5 mL LB medium containing 50 µg/mL kanamycin, 7 µg/mL gentamicin, and 10 µg/mL tetracycline. Incubate at 37 °C with 250 rpm shaking for 16 h.
   4. Isolate the recombinant bacmid DNA harboring the tag-K-bZIP, quantify, and re-suspend at a concentration of 1 µg/µL.
3. Generate recombinant baculoviruses expressing tag-K-bZIP by transfecting 1 µg of recombinant bacmid DNA containing tag-K-bZIP into Sf9 cells in one well of a 6-well plate.
   1. Seed 2 x 10⁶ Sf9 cells (see Table of Materials) in 2 mL Grace'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 °C for 1 h.
   2. Maintain a log phase culture of Sf9 cells in Grace's Insect Medium supplied with 10% FBS, 1% gentamicin, and 1% detergent C (see Table of Materials) at 27 °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).
   3. Add 2 µL bacmid DNA (1 µg/µL) and 98 µL reduced serum media (see Table of Materials) into a 2 mL tube. Then, add 4 µL transfection reagent (see Table of Materials) to the tube and mix well. Incubate at room temperature for 15 min.
   4. Add this transfection mixture (104 µL) into each well, and incubate at 27 °C for 12-16 h.
   5. Remove exhausted media with gentle aspiration, and replace with 2 mL of fresh Grace'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.
4. Collect recombinant baculovirus after transfection and amplify baculovirus to obtain high-titer stocks (P1) for further experiments.
   1. 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.
   2. Centrifuge at 4 °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 °C.
   3. Seed 1 x 10⁷ Sf9 cells in 10 mL Grace's Insect Medium supplied with 10% FBS and 1% gentamicin in a 10 cm Petri dish and incubate at 27 °C for 1 h.
   4. For amplification of recombinant baculoviruses expressing tag-K-bZIP, add 0.5 mL P0 baculovirus to the seeded Sf9 cells and incubate at 27 °C for 48 h.
   5. Collect supernatant as P1 baculovirus in a 15 mL tube and store at 4 °C for up to 1 month.

2. Purification of K-bZIP

1. Maintain a log phase culture of Sf9 cells in Grace's Insect Medium supplied with 10% FBS, 1% gentamicin, and 1% detergent C at 27 °C in orbital suspension at 140 rpm as described in step 1.3.2.
2. 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.
3. Incubate Sf9 cells for 72 h at 27 °C on an orbital shaker with 140 rpm shaking, then collect by centrifugation at 2,740 x g for 15 min at 4 °C.
4. 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 °C for 30 min.
5. Centrifuge cell lysate at 15,000 x g for 15 min to collect the supernatant.
6. To purify tag-K-bZIP, incubate the cell lysates (10 mL) with 50 µL of antibody-tagged magnetic beads in 14 mL polypropylene tubes, and rotate at 50 rpm for 3 h at 4 °C using a suspension mixer. Following protein capture, pellet the beads at 800 x g centrifugation for 30 s at 4 °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.
7. 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.
8. Wash the beads with lysis buffer four times.
   1. 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.
   2. Repeat the previous step another 3 times.
9. Wash the beads with phosphate-buffered saline (PBS) once.
   1. 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.
   2. Put the 1.5 mL tube on a magnetic stand and then remove the supernatant.
10. Elute tag-K-bZIP protein from the antibody-tagged magnetic beads by adding 100 µL of 150 µg/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.
11. Analyze 1-5 µL purified tag-K-bZIP protein by SDS-PAGE followed by Coomassie blue staining. Load 2 µg, 1 µg, and 0.5 µg 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.
12. 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/µL and store in small aliquots at -80 °C.

3. SUMOylation Assay

1. Add 3 µL of 100 ng/µL of purified tag-K-bZIP into the master mix of each in vitro SUMOylation reaction. Components in master mix contain 1 µL protein buffer, 1x SUMOylation buffer, 0.5 µL p53 protein (0.5 mg/mL), 25 nM of E1 activating enzyme (stock conc.: 1 µM), 50 nM of E2 conjugating enzyme (stock conc.: 10 µM), and 250 nM each of the SUMO peptides (SUMO-1, SUMO-2, or SUMO-3; stock conc.: 5 µM) to a final volume of 17 µL.
2. Mix the contents gently and incubate the reaction at 30 °C for 3 h. Stop the reaction by adding 20 µL 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 β-mercaptoethanol) and denature the samples at 95 °C for 5 min.
3. 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.
   1. Set up the gel apparatus and load 20 µL of sample into gel wells.
   2. 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.
   3. 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.
   4. 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.
   5. Remove the safety cover of the semi-dry electrophoretic apparatus.
   6. Pre-wet filter paper, and prepare a gel sandwich on the bottom platinum anode as follows: filter paper, PVDF membrane, gel, and filter paper.
   7. 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.
   8. 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.
   9. Hybridize the PVDF membrane with anti-p53 antibody diluted (1:1,000) in blocking buffer for 12-16 h at 4 °C with 30 rpm shaking using a suspension mixer.
   10. Take out the PVDF membrane into a container and put in TBST. Rinse PVDF membrane with TBST twice more.
   11. Soak the PVDF membrane in TBST for 30 min with 45 rpm shaking by orbital shaker.
   12. 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.
   13. Wash the PVDF membrane with TBST 3 times as described in step 3.4.10.
   14. 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 °C for up to 12 h.
   15. 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 µL 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.
   16. Expose the blot using a luminescence imaging system or autoradiography film.