A super basic cell biology question for cell biology people. I will try to measure protein phosphorylation, and I will do western blot to see whether the phosphorylation occurs. If phosphorylation occurs, does the band have to look stronger or weaker as compared to control (no phosphorylation)?
Thanks in advance.
Hi Froylan,
To measure protein phosphorylation, you will need an antibody that specifically targets the phosphorylated residue. So, you will only detect the fraction of the proteins that are activated and, in your control, you will see the levels of basal activation (which in many cases is no band at all).
That way, you cannot make a direct comparison between the phosphorylated and the total protein bands, it is like measuring a different proteins.
I hope this help,
Good luck!
Hi,
I’m quoting from [Kevin A Janes. An Analysis of Critical Factors for Quantitative Immunoblotting. Sci Signal. 2015. 8(371):rs2. doi: 10.1126/scisignal.2005966]:
“To avoid the need to strip and reprobe the same blot, an alternative is to use two-color fluorescence detection with phosphorylation-specific and total antibodies that are raised in different hosts and against different epitopes (Fig. 7C).”
Thanks Priscila Marcondes - it is really useful your comment to me. I really appreciate.
Whether the band appears stronger or weaker or the same in western blotting would very much depend on the specific antibody, whether its interaction with the antigen is affected by phosphorylation. However, very frequently in SDS electrophoresis the electrophoretic mobility is also affected by phosphorylation, and you may be able to distinguish two bands for the phosphorylated and non-phosphorylated species when probing with a phosphorylation-independent antibody.
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