Hi All,
I am facing some problem with beta actin as loading control in myogenesis western blots (not getting normalized). So is it obvious problem with beta actin or i should use another loading control for myogenesis western blots??
Please suggest.
Thanks in advance!!
Hello Utsav.
beta-Actin should serve as a loading control in any cells including differentiating myoblasts or other phenotypes. Your protein extraction from the differentiating myoblasts, myotubes or muscle fiber needs more careful attention to dissolve proteins from the moment of cell lysis and protein dissolution in SDS-sample buffer. I suspect incomplete protein extraction.
If you use mortar and pestle to grind the cells or tissues after plunging in LN2 gas, followed by lysis of the ground cell powder and SDS-sample buffer, it never fails for tough tissues with lots of cytoskeletal and its associated protein extraction.
Best wishes.
Hi, Utsav,
I tried to use beta actin but, in some experiments, it did not work well (lineage C2C12).
An option that worked well was the GAPDH and also perform normalization by Ponceau.
Any doubts I am available,
Good luck!
Hi,
The journal of biological chemistry (JBC) declares in the editorial recommendations that "Normalize signal intensity to total protein loading (assessed by staining membranes for total protein) whenever possible. “House-keeping” proteins should not be used for normalization without evidence that manipulations do not affect expression.".
For more information the JBC indicates:
http://jbcmission.asbmb.org/collecting-and-presenting-data
I suggest as total protein quantification method the membrane staining with Ponceau S., Coomassie Blue or Amido black.
Best regards.
Mauricio
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