Hi Rizwanna,

Skeletal muscle can be rapidly separated from surrounding tissues, quickly snap-frozen in liquid nitrogen and then can be powdered under liquid nitrogen in an ice-cold mortar & pestle. The powdered skeletal muscles can be then homogenised (dounce homogeniser, 5-7 strokes) in ice-cold lysis buffer [50 mM Tris, pH 6.8, containing 2% SDS ] supplemented with protease inhibitors. I did not find RIPA buffer (ThermoFisher Scientific) as effective as SDS-based buffer while extracting proteins from rat skeletal muscles.

It is also advisable to fractionate your samples (cytosolic or nuclear) and use the appropriate fraction for your protein of interest. You get a cleaner result at the end in Western Blot analysis.

The homogenates can then be further sonicated (60 sonic, Dismembrator; Fisher Scientific, Pittsburgh, PA) using two 50-s cycles of 5–7 W. Centrifuge the resulting suspensions at 13,000 rpm for 10 min at 4°C and collect the supernatants. The protein content of the supernatant can be determined using BCA (Thermo Scientific) and subject 50 μg of protein to Western blot analysis. Follow the manufacturer's protocol to perform the BCA assay using BSA as your standard protein. Your plate-reader will create the standard curve (using the equation Y=mX+C generated by plotting the std conc of BSA in X axis and absorbance or OD in Y axis) and estimate your protein concentration in the sample.

Good Luck

Thank you for your answer, its helpful. Do you have any prorocol for BCA Assay?

I have used this one for skeletal muscle. As for what you use for the lysis buffer this will depend on what proteins you are investigating. I have used RIPA, or an NP-40 based one. If you use SDS don't go over 2% as it will interfere with the BCA assay. https://www.thermofisher.com/order/catalog/product/23225. Here is a compatibility chart for BCA assay and lysis components https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0068-Protein-assay-compatibility.pdf