I am trying to detect PIkBalpha on my blot. The Primary and secondary antibody dilutions prepared were absolutely fresh. O/N incubation with primary antibody after Ihr RT blocking and 1 hr incubation with secondary antibody eventually. When I developed the blot, I saw no bands and afterwards I stained the blot with Ponceau to check the transfer(eventhough the marker transferred fine), I could confirm that my blot has the proteins. Why doesn't it work ?
How many washes you did and for how long ?
Have you added positive control in order to see if your antibodies work ?
Two washes over a span of 15 mins after the primary antibody incubation and two washes over a span of 30 mins (constantly shaking on the shaker) after the secondary antibody incubation. There is no positive control to check the activity of the primary antibody but I literally opened a fresh vial and prepared the fresh 1:1000 dilution, right before use, to confirm the primary antibody is not degraded.
Positive control is important in order to have a proper idea of what went wrong. Now we dont know whether your protein is not displaying tag or whether it is something you missed during blotting so that you dont have signal. Sometimes if you use less dilution and if you wash secondary for long, you wont see signal then you can incubate blot again with the secondary ab and later wash 3 times for 5 min.
You might be right, I did see very very faint bands on longer exposure. Now I am wondering if this is because either of the two antibodies did not work, or the protein(40kDa, small) did not get transferred properly or overall the amount of lysate loaded on the gel is low(which is unlikely because the Ponceau confirms enough protein on the blot).
I just confirmed the loading control (Actin 42 KDa), so the developing solutions and the wash buffers are fine/ not contaminated with azide. Now, it could either be that proteins smaller than 42 KDa which includes my PIkbalpha at 40 KDa do not get transferred to the blot, or either of the two antibodies are not working.
I also confirmed the activity of the anti rabbit IgG HRP linked secondary antibody on a different blot for the detection of a different protein which probably confirms that the secondary antibody is not at fault here. The primary antibody seems to be the main suspect, if I am not missing anything but I dont understand how that could be as the dilution was freshly prepared.
42 and 40 KDa is not great difference. Rather than working backwards and keeping poncaue as your best bet, if you calculated protein concentration then you know how much you loaded, and taking other blot as a standard to guess what was wrong with the earlier is not good. You told that you can see the protein after exposing long which leads to the loading problem because 1:1000 of fresh primary ab vial is more than enough, sometimes if you load enough even 1:10,000 gives signal. If you can then please load more, add control and repeat with less washing - 5 min 4 times.
You were right Mahesh, the Antibody dilutions work rather well for the same amount of lysates loaded from a different cell line. Apparently the cells I was using do not produce as much antigen anymore, I think this could be on account of the cells being in culture for too long(because in the past they have worked well). My current opinion is that I would have to use freshly thawed cells.
Yeah, thats what it looked like for your issues. I dont know which cell line you are using to quote maximum passage no, but several passage is not ideal. Sometimes WB will take few steps to optimize, good to know that you finished troubleshooting.
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