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