5 replies to this topic

[issactay]

hi,i'm doing western blot for beta-catenin and APC.i can't get bands for both protein.my samples were extracted from FFPE tissues.Any suggestions?what is the gel percentage i should use as well.thank you!!!

[casandra]

issactay, on 21 May 2011 - 08:03 PM, said:

hi,i'm doing western blot for beta-catenin and APC.i can't get bands for both protein.my samples were extracted from FFPE tissues.Any suggestions?what is the gel percentage i should use as well.thank you!!!

Hi issactay and welcome to the Bioforum. It would help if you can provide more details about your western experiments eg extraction method, lysis buffer, size of your target proteins,  running and transfer conditions etc. Normally, the smaller your protein is (with low molecular weight)the higher the percentage of gel is recommended. Abcam has a good western blotting guide as well as trouble-shooting tips...the Roche handbook as well...you can also check this- our protocol online protocols .......and if it's the first time you're doing PAGE and western blotting, it's always better that you first observe your supervisor or someone else do it, and then you can try it on your own cos there could be a lot of things that can go wrong....meanwhile, read up on it too...

[Biouday]

In addition to casandra comment, you can also try to run positive control(some time it comes with antibody kit or you can ask your co worker who already working with this proteins )

[issactay]

thanks for the comments.i'm not first time doing western blot.i did many times on other targeted proteins and all works.just i can't get for beta-cat and APC.the size for beta-cat is 88 kDa whereas APC is 310kDa.for the beta-cat,a very faint band can be observed but when i repeated it,i can't get the band anymore.For APC, i tried using 8%,7.5%,6% and 5% gel before.all not work.regarding the extraction method and lysis buffer,sorry that i cannot tell here as i developed the method and lysis buffer on my own and it still confidential.any suggestions?thanks!!!

[issactay]

i using positive control for beta-cat and APC to run for the western blot.unfortunately,there is no bands.

[Adrian K]

Positive control no bands... I suggest bad/degraded/mislabeled/wrong antibody?
Also, both the % of PAGE gel and the Bis-Acryl:Acrylamide ratio is important.
Maybe your PAGE gel has higher B:A which caused the separation not good enough.
You have to play with both for an optimum separation.

I not sure if your lysis buffer consist of protease inhibitors. It might be a good idea if you can add some in it.

IF all fail, refer to this article:
http://carcin.oxford...21/11/1935.full

Try to contact the authors and see their opinion. They had done extensive and comprehensive work in both protein you mentioned.

In the mean time, keep us update about your troubleshooting and perhaps others can be benefited from your findings.