I am trying to modify a protocol to extract Myosin from muscle tissues
by cutting out the extraction portion, homogenize directly in the sample
buffer and load onto gel for visual, then do western and Ab staining. I
have SDS in the sample buffer along with BME, Tris, and Protease
inhibitors, ATP, and Na Vanadate. I am getting clear bands on the gel,
but no band where the Myosin should be. I keep the entire solution cold
(on ice) until I spin and remove supe that is then boiled and loaded. I
was getting quite a smear initially, but has improved following an
additional spin, after boiling. Still no Myosin. I guess it is not
coming out of the tissue. I assume I need to bring the buffer up to an
appropriate temp for the SDS to be effective.
The other part of this problem is I cannot easily quantitate with
Bradford or Lowry method in this buffer due to either the SDS (Bradford)
or the BME (Lowry). I can leave out the BME during homog then take out
aliquot to assay and add BME to remainder, but am not sure if the BME
should be in there during the homog. Is there another way of
quantitating (maybe after run onto gel) that would be easier to get
around this problem?
I would appreciate any thoughts on these issues.
Rick
