Dear all,
I'm going to perform a western blot analysis on cell lysates from human macrophages. I have a limited amount of lysate available for analysis. I would like to know if it is acceptable to load 5 µg of protein instead of the usual 10 µg to detect MAPK signaling proteins (such as p38, ERK, JNK)? (1mm and 10 wells gel)
This should be possible, but of course is also dependent on how good your antibodies are and your macrophages eg. drug treatments.
Hello Alireza Ahmadi,
There is no general rule as such for the protein concentration to be loaded for Western Blot analysis. It mainly depends on the abundance of the antigen you want to detect, and on the affinity of the primary antibody. If your antigen is highly expressed and if you have a good antibody, you can go down to less than 5µg total protein.
For instance, the growth and survival of cancer cells is often driven by constitutive activity in the MAPK and phospho-inositide 3-kinase (PI3K)/AKT signaling pathway.
In such constitutively active cell lines, 5 to 30µg of total protein is sufficient for the detection of ERK phosphorylation. I am not aware of human macrophages. You may/may not get the signal depending on factors which Diana Baxter has already mentioned.
Best,
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