Hi everyone, I've tried western 5 times. But I can't get a picture.
I loaded my proteins in 30 microliters, ladder 5 microliters. They progressed very well. I performed the transfer phase on ice. I added the methanol of the transfer buffer afterwards. I stained it with Ponceau s and it looked good. I incubated the primary antibody overnight in the shaker. I did the washes with TBST. I added secondary antibody. It spun in the shaker for 2 hours. I added the latest hrp solution and went to view. but i didn't get a picture. What should I do ? What is the problem ?
Did you wash your membrane with TBS-T after 2ndary antibody incubation?
I am a little confused by your statement "I added the methanol of the transfer buffer afterwards." Do you mean that your transfer solution did not have methanol in it when you did the transfer? Because if that's the case, the protein wouldn't transfer properly.
Also, do you know for certain that the antibodies you have work for the protein you are trying to look at? I'm not seeing any bands at all, which suggests to me that either your primary isn't binding to your protein or your secondary isn't binding to your primary. You may want check with a primary that you know works and make sure that you are using a secondary from a species that will react with your primary (for example, if your primary was made in mouse you would want a secondary that is anti-mouse). Furthermore, you didn't mention your blocking step. Is it possible that you over blocked your membrane and accidently prevented your primary from binding to your protein of interest?
Tricia Gilliland , there was methanol in the transfer .I didn't add it after the transfer. The antibodies I use are also correct. I'm just not sure about the substrate.
Sude Naz Çataltepe What is it about the substrate that has you concerned? Have you tried staining for a housekeeping protein like GAPDH or b-actin and gotten good results? If you're confident in your antibodies and other proteins show up well, it may be that your sample doesn't express the protein you're looking for. Without knowing what antibodies you're using nor what tissue you're testing on, I can only guess that part. On the other hand, if the sample isn't showing proteins that should be there, there may be an issue with sample itself. For example, if it was left out for too long and the proteins degraded you may still see something with Ponceau s but the epitopes may be degraded to the point where the antibody no longer recognizes them. Or the problem could be the lysis buffer you are homogenizing your samples in, or if you forgot to add PMSF to your lysis buffer.
You may need to look at your whole protocol starting from sample collection to solve this.
Tricia Gilliland First of all, thank you for your valuable comment. I solved my problem. There was a problem with the substrate. (Horseradish peroxidase solution). It fixed itself when I tried another substrate. And I was able to get my image.
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