Akt/PKB kinase assay

 

  1. Preincubate tubes containing 15ml of 50% protein G sepharose and 5ml Akt-1 antibody (C-20, Santa Cruz) or 3ml Akt-1 antibody (UBI #06-558) in 200ml Akt buffer for 1 hour.
  2. Spin down the gel and aspirate the supernatant.
  3. Add 1-1.5mg protein lysates (Akt lysis buffer)
  4. Incubate 1-2 hrs. at 4C
  5. Wash the gel with 3x 1ml Akt lysis buffer and 2x 1ml kinase buffer
  6. Aspirate the remaining buffer completely
  7. Add 20ml of 1.5 Akt kinase reaction buffer
  8. Add 10ml of start solution
  9. Vortex and incubate @RT for 15 minutes on shaker
  10. Stop the reaction by adding 10ml of 5x SDS sample buffer and boil for 5 minutes
  11.  Apply 20ml of recction on 15% SDS-PAGE
  12. Transfer or dry up
  13. Autoradiography

 

 

 

Akt lysis buffer:                                                    Kinase Buffer

20mM Tris-HCl (pH 7.4)                      20mM Tris-HCl (pH 7.4)

                              5mM EDTA                                         10mM MgCl2

                              10mM Na4P2O7                                               1mM DTT

                              100mM NaF                                        2mM Na3VO4

                              2mM Na3VO4

                              1% NP-40

                              1mM PMSF

                              10mg/ml aprotinin

 

1.5x Akt kinase reaction buffer

                              75mM Tris-HCl (pH 7.4)

                              15mM Mg Cl2

                              1.5mM DTT

                              1.5mM protein kinase inhibitor

                              1.5mg/ml BSA

 

Start solution

                              50mM cold ATP

                              3mCi g-32P ATP

                              0.2mg/ml histon H2B (Roche #223514 $120)

Lipofectamine PLUS

10cm plate

 

  1. 50-90% confluent cells
  2. pre-complex the DNA with the PLUS reagent; 4mg DNA + 750 ml serum-free media-mix; add 20ml PLUS reagent-mix ;incubate 15 minutes
  3.  Dilute Lipofectamine reagent 30ml + 750 ml serum-free medium-mix
  4.  Combine 2+3-mix; incubate 15 minutes
  5.  Replace the medium with 2.0ml serum-free medium
  6. Add 4 complex to cells-mix; incubate 3 hours
  7. Add complete media +1mL FBS
  8. Change the media on the day after transfection
  9. Harvest cells 24-48 hrs after transfection

CDNA synthesis (Roche #1483?88)

10x reaction buffer              2ml

25mM MgCl2                    4ml

deoxynucleotide                  2ml

OligoDT or random primer 2ml

Rnase inhibitor                    1ml

AMVreverse transcriptase  0.8ml

Gelatin                                0.4ml

RNA                                  1mg of total RNA

H2O                                  to total 20ml

 

  1. Vortex and spin down
  2. 25C for 10 minutes (annealing)
  3. 42 C for 60 minutes
  4. boil for 5 minutes
  5. on ice 5 minutes
  6. keep @ -20 C
  7. use 2ml for RT-PCR