Application
Western blot
Sample
Human Cell lysate - whole cell (HeLa cells)
Loading amount
100 µg
Specification
HeLa cells
Treatment
10 µM MG-132 for 6hrs
Gel Running Conditions
Reduced Denaturing (10% Tris-Gly gel)
Blocking step
LI-COR® Odyssey® Blocking Buffer as blocking agent for 45 minute(s) · Concentration: 50% · Temperature: RT°C
Other product details
Dilution
1/2000
Incubation time
14 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 50mMTris,pH8.0,50mMKCl,1mMEDTA,5%glycero
Secondary antibody
Name
Non-Abcam antibody was used: AlexaFluor 680 goat anti-mouse
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor® 680
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor® 680
Dilution
1/10000
Detection
Detection method
Odyssey® scanner
Exposure
3 minute(s) and 0 second(s)
Bands
Specific: above 150 kDa Non-specific: 50, 115, 145 kDa
Positive control
HeLa cells treated with MG-132
Negative control
HeLa Whole Cell Extract
Additional data
Additional Notes
Figure legend.
HeLa cells were cotransfected with a plasmid expressing a target protein together with Ubi expressing vector for 24 hours and either left untreated (Contr) or were treated with 10 µM MG-132 for 6hrs (+MG132). Then the protein of interest was pulled down using Flag agarose beads and and probed with Ubi-1 antibodies. The protein is known to be degraded through proteasome.
HeLa cells were cotransfected with a plasmid expressing a target protein together with Ubi expressing vector for 24 hours and either left untreated (Contr) or were treated with 10 µM MG-132 for 6hrs (+MG132). Then the protein of interest was pulled down using Flag agarose beads and and probed with Ubi-1 antibodies. The protein is known to be degraded through proteasome.
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提交于 Dec 13 2011