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

Samenvatting

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

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....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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