I am wanting to detect human keratins by Western blotting and look for changes in levels of each keratin. I wanted to find out what the best pan-keratin antibody is available to quantify changes in specific forms before proceeding to order keratin-specific antibodies. Also, can anyone tell me if skim-milk powder contains keratin (troubleshoot blocking step as currently using 5% BSA)?
I remember that AE1 and AE3 could be dirty when being detected by Western blot, but they are ok to start with; I used them for IF. My experience in the past was to perform a cytoskeletal extraction before proceeding further, It should be cleaner! I would include fibroblasts as a negative control in all the experiments as well. You can refer to my paper in Cancer Letters on the immortalization of an esophageal epithelial cell line (Cheung et al. 2010). I remember I started with purifying the cytoskeletal fraction, detect the keratin patterns by SDS-PAGE followed by Coomassie Blue staining of the gel. If the separation is good, you can actually kind of estimate the presence of specific keratin(s) based on the size and whether they are the 'right keratins' in the tissue type you are working on. You can take a look at this old paper:
R. Moll, W.W. Franke, D.L. Schiller, B. Geiger, R. Krepler, The catalog of human cytokeratins – Patterns of expression in normal epithelia, tumors and cultured-cells, Cell 31 (1982) 11–24.
Also be careful in the amount of protein you load to the gel for analysis, Western Blot is super sensitive and keratins are abundant, optimizing the amount of protein loading can help give you a cleaner preliminary answer.
For your second question,
If I remember correctly, I didn't change the milk-blocking to BSA, I think it should be fine!
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