SDS will percipatate out if cold. You might try the lithium salt of
laural sulfate which IS cold soluble, albeit somewhat expensive (Sigma
carries it still I think). But with all those inhibitors in the
solution, why not just homogenize and boil the extract before
centrifugation?
To quantitate, you might try taking an OD280. It's a bit old fashion,
but it it works !?!
Jim Kami
Blue Rose Biotech
Rick Bright wrote:
>> 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
