My protocol: lysate in RIPA buffer; 1D in pre-cast 4-12% gradient gel; transfer at 30V over 2 h; block in 8% non-fat milk in tris-buffered saline with 0.1% tween 20 (TBS-T); incubate with primary at 4c overnight in TBS-T; 3 washes of 10 min each; 1 h in Alexa fluor secondary at room temp in TBS-T; 3 washes of 10 min each; scan.
Try using the lysis buffer of this article:
http://www.molbiolcell.org/content/22/8/1409.full.pdf+html
Here you have not mentioned whether you r getting non- specific background or no signal at all. in case of no signal try using 5% BSA as blocking agent ,as milk is more stringent blocker or u can also try decreasing the primary antibody dilution.
@Ankit- There are no bands at all for either of the two proteins...It's probably not fair to club the two proteins into one question 'cause they could be a no-show for different reasons but I'll try the BSA. Thanks.
Modify the following
1. Try nitrocellulose membrane
2. Transfer at 100V for 2 to 2:30 hrs. [30v for 2hr is definitely not sufficient for PKCs]
3. Block in 5% non-fat milk in TBS-T (for 30 mins) and then switch to primary antibody O/N at 4 degree C
4. Prepare primary Abs in 5% milk in TBS-T
5. Check you're using right secondary Abs (i.e.. Anti-rabbit for rabbit antibody OR anti-mouse for Mouse MAbs).
6. Try to use a positive control to make sure your cell line express it or not.
@Bhumika : one more thing i would like to suggest is that RIPA is rather a very strong lysis buffer , u can try using other lysis buffers such as MKK lysis buffer or passive lysis buffer (promega). All the best.
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