We have tried various modifications to our standard Western Blotting procedures now trying to detect CHOP (GADD153) in our human cell lysates after e.g. stimulation with 1 µg/l tunicamycine, but to no avail.
Do you have some helpful pointers as to how to detect CHOP?
We have used nitrocellulose and PVDF membranes in the past. Primary antibody is the recommended Santa Cruz monoclonal mouse anti-GADD153 (B-3) at 1:250. For technical reasons, we don't do the transfer on ice, however, actin signal is always strong on our membranes.
Is it a matter of amount of protein loaded? We have done roughly 20-30 µg/lane so far.
Thanks for any good ideas or working protocols!
We successfully detected CHOP (GADD 153) protein with Santa Cruz rabbit anti-GADD 153 (F-168), by standard WB condition (nitrocellulose) in both mouse cells and tissues. You should try to increase the amoun of protein in my opinion. How long did you stimulate the cells?
Best regards
You could try an IP (immunoprecipitation). This is what we do if we cannot detect a Protein in wb.
Protocol:
2x106 differentiated APCs were harvested (centrifuged 10min, 1300 rpm, 4°C) for each experimental condition, washed twice with ice cold PBS. Cell pellets were resuspended in 50 µl of RIPA buffer and lysed on ice for 30 min with frequent vortexing. After sedimentation and removal of cell fragments for 20 min at 10,000 rpm lysates can be used for IP (after adjusting Protein concentration).
For Immunoprecipitation lysates were incubated with protein A-coupled Sepharose beads (25μl beads e.g. Sigma Aldrich + 5μg antibody) overnight on a shaker at 4°C and then centrifuged at 14,000rmp for 15 secs on the next day. The supernatant is then removed and stored for further analysis of other proteins (e.g. Actin as control) of interest whilst the cell pellet of the current precipitated protein of interest was washed 3 times (1x RIPA buffer, 2x TNE buffer). After washing the pellet was re-suspended in 25μlRIPA buffer and 5μl SDS and cooked for 3 min at 100°C before being loaded onto SDS-Page and ready for electrophoresis.
TNE buffer
50 mM Tris–HCl (pH 7.4), 100 mM NaCl, 0.1 mM EDTA
Thanks for the advice. The timepoints we've used most consistently were between 6-8 hours. I think we will try and get higher concentrated lysates.
Hi Christian,
we tested our cells for protein expression after 18 hours of stimulation or even longer in mice. After 6 hours incubation we get increase in mRNA expression of CHOP.
For reference
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0068017
Regards
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