Protein extraction from rat liver tissue - (Feb/24/2005 )
I'm looking for a protocol to extract proteins from rat liver tissues. Then I will detect the level of a membrane bound tyrosine kinase receptor with western blotting.
I found some protocols for protein extraction (the one with NP-40 and the one with Camiolo buffer). But I'm not sure if these methods can be efficient for extraction of transmembrane proteins.
Is there anyone who could offer a suitable protocol?
thanks a lot...
For liver microsomal membrane bound and cytosolic proteins I just did the following:
Livers were weighed and three volumes of 67 mM phosphate buffer containing 1.15 % KCl added to give a final homogenate concentration of 25 % w/v before they were homogenised at 8000 rev.min-1. To ensure all tissue was completely homogenised, after the initial homogenisation, samples were placed in a glass homogenising tube and ground further by hand. The homogenate was transferred to centrifuge tubes, the tubes balanced and the samples centrifuged at 10,000 ´ g for 20 minutes at 5 °C (Sorvall RT7 Plus). The supernatant (S9) was removed with a clean transfer pipette and transferred to a Beckman ultracentrifuge tube, accurately balanced and capped. These tubes were placed in a pre-cooled rotor (4°C) and centrifuged at 100,000 ´ g for 1 hour (Sorvall S90). Supernatant (cytosol) was removed and stored in aliquots at –80 °C until use. The microsomal pellet was rinsed carefully with 67 mM phosphate buffer containing 1.15 % KCl and the washings discarded. 1 mL of the buffer was added, the pellet teased away from the side of the tube, rinsed into a microcentrifuge tube then resuspended in a small volume (2-5 mL). The microsomal suspension was stored in aliquots at –80 °C until use.
Hepatic microsomal samples were diluted to 1 mg.mL-1 with MilliQ water and then further diluted 1:4 (final protein concentration 0.25 mg.mL-1) in sample buffer (0.5 M Tris-HCl, pH 6.8 containing 2 % sodium dodecyl sulfate (SDS), 5 % glycerol, 0.1 M DTT and several grains of bromophenol blue).
Worked fine for five years for me!