Sean Lauber:Generating Cell/Tissue Lysates

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Note that this is for tissue homogenates (lung) but can easily be applied for cell lysate generation. Use less RIPA and use a cell scraper to collect cells, then pass through a syringe to lyse.

1) Add the frozen lungs to 1 ml of RIPA buffer in a round bottom tube

 To prepare 10 ml RIPA:
   1x PBS = 9.3 ml PBS
   1.0% Igepal CA-630 = 0.1 ml 
   0.5% sodium desoxycholate = 0.5 ml of 10% sodium desoxycholate
   0.1% SDS = 0.1 ml of 10% SDS
 Then add inhibitors (fresh every time) for 1 ml RIPA:
   1.0 mM Na3VO4 = 5 ul of 200 um Na3VO4 stock
   1.0 mM PMSF = 5 ul of 20 mg/ml PMSF stock
   5.0 ug/ml Aprotinin = 30 ul of aprotinin stock
   1.0 mM DTT = 1 ul of 1.0 M DTT

2) Prepare one tube with 40 ml of PBS and two tubes with 40 ml of 70% EtOH

3) Prior to use, wash the homogenizer by dunking in the first 70% EtOH then in the second, then in PBS, with pulsing each time

4) Homogenize tissue samples at 3.5 - 4 for about 5 seconds, then turn off to allow for cool down, then repeat 2-3 more times

5) Clean the homogenizer as in step 3

6) Keep your samples on ice and when you're done clean up the homogenizer

7) Centrifuge at 12 K rpm for 10 min at 4*C

8) Collect the supernatants into new tubes and store at -80*C