Hello, I have started working with Western blot recently and have difficulties getting the beta-actin band. I recently tried the steps for getting a beta-actin band from the intestinal sample of mice. I mentioned it here.
1- 10μl or a maximum of 20μl of protein into each well. Start at 100 V for 1-1:30 h.
2- Transfer protein from gel to membrane (make sandwich) 100V, 1:30h
• PDVF membrane; 1 min in methanol 100% for activating. However, I only get bands on the membrane after staining but no bands after the antibody-blocking steps.
I would be so happy if you could suggest me t how I can solve this problem.
What are the duration and conditions of primary antibody incubation? and how about the validity of primary antibody?
and how about secondary antibody and detection methods?
the problems may be in these steps.
best regards
When you say "bands", are you talking about the bands you see after Ponceau as in your picture, or are you talking about the bands you should see after detection methods?
i use the following procedure and get good bands,
Protein sample: I load almost 10Ul where as the protein concentration is about 14-17ug/10ul
Gel%: 10% for beta-actin is good
Electrophoresis: For upper gel i use 65 for 30 mins to bring all samples to a level and then change to 100-110 for 1:20 hours.
Membrane transfer: i use PVDF 0.20um and i use HPLC grade methanol or ethanol for 5 mins for activating membrane. And after making good sandwich, no bubbles and make sure that its tightly packed (very important), i use 275 for 1:40hour or 200 for 3 hours. make sure that you put enough cooling around it so high temperature don't mess up.
Why are you using Ponceau?
1: By the looks of your picture it either the protein concentration is very low
2: the sandwich is not packed properly.
3: The anti body has some issue.
you told the volume but not the concentration. and share the band pictures what you are getting after incubations
Here are some simple troubleshooting steps for your Western blot issues with beta-actin:
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