Myofilament
Myofilament from Frozen Tissue
1)Weigh one Eppendorf tube per sample and mark its weight (this will be used later)
2)Prepare solutions. Make 9M Urea (2.7g of Urea and up to 5 mL with H2O). Allow to dissolve by spinning on “hot dog roller.” Once urea is in solution (should take about 10 minutes), add 0.5 g mixed bed resin and allow to spin for 10 minutes. Filter resin out of solution using syringe.
For the tissue solutions, you need SRB (standard rigor buffer) with and without Triton-X100 (0.3% v/v). SRB solutions should be supplemented with protease and phosphatase inhibitors at 1:100 ratio (ex:100 ul per 10 mL)
3)Gather samples from the -80 freezer and keep on dry ice
4)Add 1 mL of SRB with Triton to glass culture tube
5)Cut small piece of tissue and place in tube with SRB+Triton. Return rest of sample to -80.
6)Homogenize tissue at 7500 rpm for one sec at a time. Usually takes 3-4 times to be homogenous… may take longer with some human samples
7)Centrifuge sample at 1,700 x g for 2 minutes at 4° C. Discard supernatant.
8)Wash pellet by resuspending in SRB w/o Triton, vortexing, spinning down at 1,700 x g, and discarding supernatant. Perform wash two times.
9)Weigh the tube with pellet and resuspend pellet in 9M Urea at 1:10 w/v. Vortex for 15 seconds.
10)Sonicate (cold room) 3x for 1.5 seconds each time
11)Centrifuge at 18,000 x g for 10 minutes
12)Collect supernatant (this is your MYOFILAMENT FRACTION) and discard pellet.
