Herskowitz Lab Protocol
From : Matthias Peters, 10/16/93


Making Extracts for WESTERNS

  1. Harvest 5ml cells at OD = 1.0 in 15ml falcon tube[Optional : freeze pellet in liquid nitrogen]
  2. r/s in 500ul cold TCA buffer w/ 2X Protease inhibitors
  3. prepare tubes with glassbeads and 500ul cold 20% TCA
    keep on ice
  4. put cells in TCA tubes
  5. bead-beat 2X 30" at high setting, (keep on ice inbetween)
  6. take liquid phase off with thin tip into new tube on ice N.B. don't spin yet)
  7. wash beads with 500ul 1:1 TCAbuffer/20% TCA
  8. bead-beat again 30" take liquid to eppendorf
  9. spin 10' in coldroom
  10. aspirate off s/n
  11. r/s pellet in 300ul TCA-Laemmli buffer with inhibitors
  12. boil 10'
  13. spin 10' at RT, put s/n into fresh eppendorf
  14. run 20ul (=1ug protein approx)

Solutions for WESTERNS

TCA Buffer for 10ml

200ul 1M Tris 8
66.6ul 7.5M ammonium acetate NH4OAc
40ul 0.5M EDTA (important for protease inactivation)
9.7ml H2O
+ 200ul protease inhibitors : (ie to 2X final conc)
PMSF (stocks generally 100X)
Trasylol
Benzamide

TCA-Laemmli Loading Buffer

SOL I:
3.5ml 25% SDSor 4.375ml 20%SDS
3.5ml 100% glycerol 3.5 ml 100% glycerol
1.0ml 1M Tris base (not pHed)
spatula tip of bromophenolblue
-> H2O to 12ml 3.125ml H2O

SOL II:
200mM Tris Base(not pHed) -- 2ml of 1M
20mM EDTA -- 0.4ml of 0.5M
----> makes 10ml

SOL III:
1M DTT in H2O stored at -20 (1.543g/10ml)
mix:
480ul SOLI
400ul SOLII
120ul SOLIII --> gives TCA Laemmli buffer


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