1)In the cold room,wash cells with cold "Tris" (the same stuff used for TC).

Spin down the cells if you are working with suspension cells in a low speed swinging bucket centrifuge,resuspend in approximately 1 ml Tris and spin at 2K in a microfuge.

Wash on plate if adherent cells by aspirating medium,adding 2 to 10 ml Tris,and aspirating again.

2)Lyse cells at 107 cells/ml or (minimally)500 microliters/10 cm dish for adherent cells in lysis buffer (e.g.NP-40,RIPA).

Gently resuspend the suspension cells in the lysis buffer in the eppi tube using a plastic pasteur pipet or Pipetman.Scrape adherent cells off the dish with a rubber policeman,but leave them in the dish.

3)Incubate lysing cells for 20 min at 4℃.Scrape the lysate of adherent cells to the side and transfer to an eppi tube.

4)Clarify lysate by centrifugation in the refrigerated TOMY in the lab at 15 K rpm for 20 min at 4℃ (longer for RIPA lysis).If you are using 32P,be sure not to overfill the tube and be sure to use a screw capped tube.

5)Wash "bugs" (Staph.a)--that are stored in tube on coldroom shelf--in lysis buffer.To do this,take out a measured volume--more than you need--and mix with twice this volume of lysis buffer.Then spin down the bugs in microfuge,and resuspend in the "original measured volume" in lysis buffer.)

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You have a choice to either preconcentrate/prebind the antibody to the bugs and then add lysate to this or to mix lysate with antibody and then add the bugs.Bart prefers the latter.

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6,7)Preconcentrating Ab to bugs:

Mix bugs (20 microliters/IP)with antibody-incubate 20 min,4℃.

Add lysate-incubate 45 min,4℃.

Skip to step 8.

OR

6)Aliquot Ab in tubes.Add clarified lysate and incubate 45 min,4℃.

7)Add 20 microliters bugs/IP and incubate 20 min,4℃.

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8)Spin down bugs to which are bound the antibody and the antigen,aspirate supe,and resuspend by vortexing in 500 microliters lysis buffer.

9)Transfer to a new eppi tube and wash IP 3x with 500 microliters lysis buffer and then 1x with TN by spinning and resuspending.

10)If doing kinase assay,do additional wash in TN and then proceed with kinase assay (below).

11)Pellets can be stored dry at -20℃ for short term (one or two days)or at-70℃ for longer.

12)Resuspend pellet in at least 50 microliters sample buffer with 5% fresh b-ME,boil 2 min,microfuge 4 min,and load less than half (so you can do it again,if necessary).

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KINASE ASSAY

1)Resuspend pellets in 10 microliters of kinase buffer (40 mM PIPES,pH 7.0; 10 mM MnCl2).Keep samples on ice until use.

2)Make up a ATP mix such that you have 2 or 10 microcuries of gamma-[32P] ATP per 10 microliters KB.When using an exogenous substrate such as enolase,add the substrate to the ATP mix.

3)Add 10 microliters ATP mix to each IP,pipetting to mix,then incubate at 30℃ for 10 min.

4)Stop kinase assay by adding 1 ml of TN+ 0.01% NP-40.Microfuge 2 min,and aspirate supe by hand using a one-piece plastic pasteur/transfer pipet.

Note: If using a soluble exogenous substrate such as enolase,stop rxn by adding 20 microliters 2x SDS sample buffer.