Hello, I'm running western blot for alpha-SMA and beta-actin from the liver sample.
Incubating in reducing buffer, 80oC for 15 mins
1st antibody: alpha-SMA ab15734 (1:1000), rabbit polyclonal
beta-actin: 13E5 (Cell Signaling, 1:2000).
2nd antibody: ab6721 (1:5000)
The result included one lower-than-37kDa band and one upper, unknown band (75-100kDa) (Attached photo)
Thank you very much, Mr. Andrew :) your advice reminds me a lot about my sample preparation!
Regarding whether my band had been formerly stained, it was stained for only once :)
Unfortunately you may run into primary antibodies that produce non-specific banding patterns that change with different cell types, etc. Always be sure to include a negative control so that you know which bands are non-specific. Sometimes they are the exact size of your desired protein!
Also definitely follow Andrew's advice as far as the addition of protease inhibitors, you may very well have protein degradation.
Do you mean to say that this is one of the blots? B-actin or a-SMA?
This looks more like the bands which you see in places with excess immune cells (heavy and light antibody chains, running slightly higher when not completely reduced). But this is usually seen while using anti-mouse secondary!
How much protein is loaded?
What was the initial developed protein/antbody?
Was the blot stripped?
Best, Manoj.
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