I am trying to get a signal for phospho-myosin light chain 2 expression in breast cancer cell lines. I am using the cell signaling pMLC2 antibody and followed their dilution and incubation suggestions. But still the signal is very weak, impossible to get a decent image. I am loading 20 ug of total proteins. I am looking for a suggestion to enhance the signal, any kind of inducer that would promote the MLC2 phosphorylation etc...
Thank you !
Dear Sandra,
If you are getting a very weak signal is likely because of (1) low concentration of your protein of interest, (2) ineficient transference to the membrane (3) poor binding of your primary antibody or your secondary antibody. These are technique-related issues. Also, a biological reason might be the loss of phosphorylation due to phosphatase activity during the protein extraction or storage of protein extracts; this could be overcome by adding some phosphatase inhibitors to your cell lysis buffer, which is generally recommended when interested in phosphorylated proteins.
For the technique-related issues, you could: (1) increase the amount of protein loaded in gels, (2) increase transference time and (3) check if your antibodies are suitable for WB (not all Abs are) and if so, you could increase the concentration of the Abs and/or increase the incubation time.
Hope these comments would be useful, and good luck (usually you need some when doing WB :-)
pMLC2 is a small protein, it might be useful to use a membrane with a size of 0.1 micrometer for blotting. I would also suggest to load more total protein (in the range of 40-50 micrograms). Finally you may try to use the new ECL product from GE Healthcare called select. The signal is greatly increased compared to standard ECL but you may get non specific bands.
Good luck
J. Bonaventure
Hello Sandun, Have a look at this paper and previous articles from Sutherland and Walsh (Michael P Walsh.
They have used band shift approaches combined with immunoblotting using an anti-'total' antibody to separate and assess LC20 phosphorylation. Note that a weak signal with a phosphorylation-specific antibody may also indicate a low stoichiometry of phosphorylation at the site in question.
http://ajpcell.physiology.org/content/310/8/C681.long
Dear Sandun,
which cell lines are you using and what experiment are you conducting?
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