Application
Immunoprecipitation
Sample
Human Cell lysate - whole cell (Colon cancer cell line HCT116)
Total protein in input
30 µg
Specification
Colon cancer cell line HCT116
Immuno-precipitation step
Protein A
Other product details
Concentration
1 mg/ml
Incubation time
16 hour(s) and 0 minute(s)
Western Blot antibody
Name
Non-Abcam antibody was used: Sigma rabbit anti mouse
Host species: Rabbit
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Rabbit
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Additional data
Additional Notes
Lyse cells as for Western Blot (Tris-triton buffer)
Prepare IP buffer (20mM Tris HCl pH7.5, 10mM EDTA, 1mM EGTA,100mM NaCl, 1% Triton-X 100. To 10 ml add 100ul Protease Inhibitor cocktail (Sigma), 20ul NaF (1M), 50ul B-Glycerophosphate (1M), 100ul PMSF(0.1M), 100ul NaVO4(0.1M))
Prepare samples (100ug protein in 100ul IP buffer)
Add 1ul antibody/100ug protein incubate on ice for 4 hours.
Wash beads 3 x in IP buffer (500ul).
Add 60ul beads to each sample and incubate overnight at 4C on rotating mixer
Centrifuge tubes for 10 seconds at 13000rpm
Remove supernatant and KEEP (Unbound fraction)
Resuspend beads in 55ul 2x Loading buffer and heat at 96C for 5 minutes.
Centrifuge briefly and remove supernatant to clean tube.
Load 30ul on SDS PAGE as normal (see Western blocking and incubation steps above)
Probed Western blot with same antibody (1 in 1000/ 5%milk TBST)- not ideal but worked well with this antibody, no interference of antibody heavy or light chain with Hsp27 band.
Very strong IP bands seen.
Prepare IP buffer (20mM Tris HCl pH7.5, 10mM EDTA, 1mM EGTA,100mM NaCl, 1% Triton-X 100. To 10 ml add 100ul Protease Inhibitor cocktail (Sigma), 20ul NaF (1M), 50ul B-Glycerophosphate (1M), 100ul PMSF(0.1M), 100ul NaVO4(0.1M))
Prepare samples (100ug protein in 100ul IP buffer)
Add 1ul antibody/100ug protein incubate on ice for 4 hours.
Wash beads 3 x in IP buffer (500ul).
Add 60ul beads to each sample and incubate overnight at 4C on rotating mixer
Centrifuge tubes for 10 seconds at 13000rpm
Remove supernatant and KEEP (Unbound fraction)
Resuspend beads in 55ul 2x Loading buffer and heat at 96C for 5 minutes.
Centrifuge briefly and remove supernatant to clean tube.
Load 30ul on SDS PAGE as normal (see Western blocking and incubation steps above)
Probed Western blot with same antibody (1 in 1000/ 5%milk TBST)- not ideal but worked well with this antibody, no interference of antibody heavy or light chain with Hsp27 band.
Very strong IP bands seen.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
Abcam user community
Verified customer
投稿 Oct 09 2006