Detection of tyrosine protein kinase by immunocomplex methods

Summary

The low abundance of tyrosine protein kinase (T P K ) expression and its rapid activation and inactivation make it difficult to purify using conventional biochemical routes. The presence or absence of specific T P K and its functional status can be detected by immunocomplex protein kinase assays.

Operation method

Immunocomplex Method for Tyrosine Kinase

Move

Immunoprecipitation and autophosphorylation assay for tyrosine protein kinase of the basic program

The following steps can be performed with lymphoid or non-lymphoid cells, primary or cultured cell lines, activated or non-activated cells.

Materials (see Appendix 1 for items with V)

Cells to be tested PBS pre-cooled on ice

I X Cell Lysate with protease inhibitors (freshly prepared and kept on ice)

5 X Cell Lysate with Protease Inhibitor (for cell activation, freshly prepared and kept on ice)

Kaumas Protein Assay Reagent (Pierce) or equivalent

Polyclonal or monoclonal rabbit anti-src-associated TPK antisera (UBI, Oncogene Science or Santa Clara)
Polyclonal or monoclonal rabbit anti-src-associated TPK antibody serum (UBI, Oncogene Science or Santa Cruz Biotechnology)

Control serum (Sigma): normal rabbit serum (if polyclonal antiserum is used), or isotype IgG (if polyclonal antiserum is used).
monoclonal antiserum)

Pansorbin (10 % formalin-fixed Staphylococcus aureus; Calbiochem), or protein A/protein
G-Sepharose (Pharmacia Biotech).

Affinity-purified rabbit anti-mouse IgG (if monoclonal antibody is used; Organon Tecnica Complex).

N P-40 buffer pre-cooled on ice

Ice pre-cooled Tyrosine Protein Kinase (T P K ) buffer

l ○/mi ○ l/L A T P solution, freshly prepared

5 m C i / m l [y-32P ] A T P (3000Ci/m m o l ; D u P o n t N E N ) : Before preparing the reaction mixture
Melt it on ice one hour before preparing the reaction mixture

Ice pre-cooled modified R I P A buffer

I X S D S / Sampling Buffer

Cell scraper (for adherent cells)

Labquake Shake Table (PGCScientific)

Heating block at 100°C

W h a t m a n 3M M filter paper

NOTE : All steps, except washing, are performed on ice or in a 4°C cold room.
Collect cells that do not require activation.

For suspension cells, collect at room temperature by centrifugation at 500 g for 10 minutes, discard the supernatant, and resuspend the cells in pre-cooled PBS on ice.
The cells should be resuspended in ice pre-cooled PBS. Centrifuge and discard the supernatant as described previously. For adherent cells, centrifuge the cells at room temperature, centrifuge at 500 g, discard the supernatant, and resuspend the cells in ice-pre-chilled PBS as described above.
Wash once with 5~IO ml of ice pre-cooled PBS, spin and aspirate the PBS. Add 2 to 3 ml of ice PBS to IO7 to IO8 cells to remove the cells with a cell scraper.
Collect the cells. Centrifuge the suspended cells in the conditions as described previously. If necessary, store the cell precipitate at 70°C for a few minutes.
Cell lysates should not be frozen (step 5).

2a. Add I ml of ice-pre-chilled IX Cell Lysate containing protease inhibitors and vortex to mix.
Collect activated cells of different time course.

Option 1 Detection of phosphorylation of exogenous substrates by protein kinases Additional material
Option 2 Detection of phosphorylation of exogenous substrates by protein kinases Additional material

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