I am unable to detect Bcl-xL, survivin, and cIAP-1 protein levels in RPMI8226 and U266 cells. I used Santa Cruz antibodies in 1 in 1000 dilution. I treated cells with a drug for 24 hours, prepared whole cell extracts, and run them on a gel. Tried many times, but never succeed. Can anyone give any suggestions please?
Antibodies from Santa-Cruz are most of the time bad (some of them work good also), so try some good company, that might be the reason for your problem.
1. Make sure you have proteins on your membrane - do ponceau staining
2. If you have proteins, then the problem is most likely with antibodies (primary or secondary) or with the detection.
3. If you use HRP-conjugated secondary antibody, make up 200ul of your detection substrate and add 1-2 ul of secondary antibody to it in dark and shake. If the HRP is working and the substrate is Ok, you should see bluish light (make sure you do this in dark).
4. If your secondary antibody and detection work, then the problem coul dbe the primary antibody. Indeed the antibodies from Santa Cruz suffer from batch to batch variation. In order to test if you antibody is working you should have a positive and negative control (ie., a extracts from a cell line that expresses these proteins and another one from cells known to be devoid of the protein). Companies like Cell Signaling Technologies or Merck Millipore have more reliable antibodies.
Good luck with your studies.
Try to increase protein amount and increase primary antibody dilution..
Try antibody from different companies...
Also quite possible these cell lines have low levels of these proteins
Hi Prahlad,
I can only agree with Ehtishamul, concerning the quality of SC ABs - always check Cell Signaling first, in my experience the best ABs out there! (no conflict of interest here...).
- For BCL-XL we are using CS - #2764 - works great 1:1000
- survivin Cs - 2808 - 1:1000
- CIAP1 was a bit harder and we used either CS - 4952 or R&D Sys. AF8181.
Apart from that I can only agree with the trouble shooting tips from Afshin, but it seems like you've done westerns before so it might indeed be an AB problem.
If all doesen't work you may want to check by RT-PCR whether your genes/proteins are expressed at all - although I'd doubt that none of them is, that would be quite a coincidence and I am not sure if cells can do without suvivin..
Hope that helps. Good luck!
I agree with Thomas- Cell Signalling Technologies have great antibodies most of the time. We have used the CS Survivin ab and it works well.
Hi Prahlad,
We also use BCLxl antibody from cell signaling and cIAP1 antibody is from R&D systems. Both of them works great . Good luck!
Sathish
Thank you all for your suggestions. It seems that antibodies are the problem in my case and I should try CST or R&D antibodies.
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