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…

Açıklamalar

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

Etiketler

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Bu Makaleden Alıntı Yapın
SUMOylation Assay: An In Vitro Technique to Detect the SUMOylation Status of Substrate Proteins by Immunoblotting. J. Vis. Exp. (Pending Publication), e21222, doi: (2023).

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