SUMOylation Assay: An In Vitro Technique to Detect the SUMOylation Status of Substrate Proteins by Immunoblotting
Published: April 30, 2023
Abstract
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.
Protocol
1. Preparation of Baculovirus Expression Constructs 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 t…
Divulgations
The authors have nothing to disclose.
Materials
| pFastBac | Invitrogen | 10359-016 | dual expression baculovirus vector |
| pCR2.1-TOPO vector | Invitrogen | PCR product vector | |
| competent cell E. coli DH5α | Yeastern Biotech | FYE678-80VL | competent E. coli cells A |
| E. coli DH10Bac | Invitrogen | 10359-016 | competent E. coli cells B |
| FugeneHD | Roche | 4709705001 | transfection reagent |
| Opti-MEM | Gibco | 31985062 | reduced serum media |
| T4 Ligase | NEW England BioLabs | M0202S | |
| CpoI (RsrII) | Thermo Scientific | ER0741 | |
| Bluo-gal | Thermo Scientific | B1690 | galactosidase substrate |
| IPTG | Sigma-Aldrich | I6758-1G | |
| Grace’s Insect Medium | Gibco | 11605094 | |
| Fetal Bovine Serum | Gibco | 10082147 | |
| Gentamicin | Thermo Fisher | 15750060 | |
| Ampicillin | Sigma-Aldrich | A9393-25G | |
| Kanamycin | Sigma-Aldrich | K0254-20ML | |
| gentamicin | Gibco | 15710-064 | |
| tetracyclin | Sigma-Aldrich | 87128-25G | |
| LB Broth | Merk | 1.10285.0500 | |
| HEPES | Sigma-Aldrich | H4034-100G | |
| NaCl | Sigma-Aldrich | S9888-5KG | |
| KCl | Merk | 1.04936.1000 | |
| Na2HPO4 | Sigma-Aldrich | S5136-500G | |
| KH2PO4 | J.T.Baker | 3246-01 | |
| sodium dodecyl sulfate | Merk | 1.13760.1000 | |
| β-mercaptoethanol | Bio-Rad | 161-0710 | |
| TRIS (Base) | J.T.Baker | 4109-06 | |
| Non-fat milk | Fonterra | ||
| glycerol | J.T.Baker | 2136-01 | |
| Triton X-100 | Amresco | 0694-1L | detergent A |
| Tween 20 | Amresco | 0777-500ML | detergent B |
| Poloxamer 188 solution | Sigma-Aldrich | P5556-100ML | detergent C |
| Protease Inhibitor Cocktail Tablet | Roche | 04 693 132 001 | |
| 3x Flag peptide | Sigma | F4799 | |
| anti-FLAG m2 Magnetic beads | Sigma-Aldrich | M8823 | antibody-tagged magnetic beads |
| SUMOlink SUMO-1 Kit | Active Motif | 40120 | standard SUMOylation protocol |
| SUMOlink SUMO-2/3 Kit | Active Motif | 40220 | standard SUMOylation protocol |
| QIAquick Gel Extraction Kit | QIAGEN | 28704 | |
| QIAGEN Plasmid Mini Kit | QIAGEN | 12123 | plasmid extraction kit |
| Polypropylene tubes | Falcon | 352059 | |
| Petri Dish | Falcon | 351029 | |
| Cell lifter | Corning | CNG3008 | |
| Loading tip | Sorenson BioScience | 28480 | |
| PVDF | PerkinElmer | NEF1002 | |
| Blotting filter paper | Bio-Rad | 1703932 | |
| Mini slab gel apparatus (Bio-Rad Mini Protean II Cell) | Bio-Rad | 1658001 EDU | |
| Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell | Bio-Rad | 1703940 | |
| Pierce ECL Western Blotting | Thermo | 32106 | ECL reagent |
| suspension mixer | Digisystem laboratory instruments Inc. | SM-3000 | |
| orbital shaker | Kansin instruments Co. | OS701 | |
| ImageQuant LAS 4000 | GE Healthcare | 28955810 | |
| biomolecular imager | GE Healthcare | 28955810 | |
| Sf9 | Thermo Scientific | B82501 | |
| anti-p53 antibody | Cell Signaling | #9282 | |
| anti-rabbit antibody | GE Healthcare | NA934-1ML |
Tags
Citer Cet Article
SUMOylation Assay: An In Vitro Technique to Detect the SUMOylation Status of Substrate Proteins by Immunoblotting. J. Vis. Exp. (Pending Publication), e21222, doi: (2023).