Met Immunoprecipitation protocol

 

Buffers:

 

1x Lysis buffer  (100 ml)

0.5% NP40                              0.5 g

         25 mM Tris pH 7.5                   5 mls 0.5 M

         100 mM NaCl                           2 mls 5 M

50 mM NaF                             0.21 g

 

add 1:250 mammalian protease inhibitors just before use

        

Only for Y-Phosphorylation experiments:

Add 1:100 Phosphatase Inhibitor cocktail II

                 

 

YP-IP Wash buffer  (100 ml)

         0.1 % NP40                                      0.1 g

         25 mM Tris pH 7.5                            5 mls 0.5 M

         150 mM NaCl                                    3 mls 5 M

 

add 1:250 mammalian protease inhibitors just before use

 

Only for Y-Phosphorylation experiments:

add 1:100 Phosphatase Inhibitor cocktail II

 

 

Protocol:

           

1.     Swell some ProteinA-sepharose (Pharmacia, fridge) in a large volume of millipore water in a 15 ml conical and mix well to resuspend all the beads.

Wash the proteinA-sepharose twice with ca. 10 bed-volumes of millipore water by pelleting the beads for 2 min at 1000 rpm in the Jouan. Then add 1 bed-volume of millipore or buffer to one bed-volume of beads to get a  50% slurry.

 

2.   Place dishes with cells on ice (metal plate on ice-bucket on a rocker) and wash three times with ice-cold PBS.

 

3.   Add ice-cold Lysis buffer to the dish. Use 0.4 ml for 6-well plate wells, 0.5 ml for 6 cm dish, 1 ml for 10 cm dish and 2.5 ml for 15 cm dish. Keep rocking on ice for 15 min (make sure buffer covers the whole surface).

 

4.   Harvest the cell lysate into 1.5 eppendorf tubes on ice. Keep samples on ice throughout the procedure. Pellet any insoluble material for 3 min at top speed at 4¡C. Collect the supernatant and discard the pellet.

 

5.   Protein assay the sample(s) (usually use 3 µl lysate from a 6 cm dish).

 

6.   Distribute equal amounts of lysate (usually 300-500mg protein) into 1.5 ml eppendorf tubes and add 5 µl of C-terminal anti-met (sc-10; Santa Cruz) or 1ml of N-terminal anti-met (DO-24; Upstate). Spin the stock for 1 min at top speed before adding to the sample.

 

 

C-Terminal:

á      Add 30µl proteinA-sepharose (50% slurry) with a cut-off tip (flick well to keep beads in suspension between pipetting).

á      Incubate on a wheel in the cold-room for 2 hours.

 

N-Terminal:

á      Incubate lysate+antibody on a wheel in the cold room overnight.

á      Add 30ml proteinA-sepharose (50% slurry).

á      Incubate on a wheel in the cold room for 2 hours.

 

7.   Pellet beads for 1 minute in eppendorf centrifuges at top speed Transfer unbound into fresh tube and save on ice.

 

8.   Wash beads 3x with ice-cold YP-wash buffer. Pellet as above and remove all but 10-20 µl of the wash to prevent losing beads.

 

9.   If the IP is to be loaded on an SDS-PAGE, wash 1x with ice-cold 10 mM Tris pH 7.5 to remove detergent, leave ~20 µl buffer on the beads and add 10 µl of 5x sample buffer. (If the IP is to be used for other purposes use adequate buffer for the final wash).

 

10.         Store remaining beads in PBS + 1/1000 Thimerosal in the fridge

 

 

1  5ml of serum contains between 40 and 80mg of IgG. MW of IgG ~150kDa and the valency of IgG is 2. Hence, 5ml of antiserum can bind 80-160mg of an antigen of MW 150Kda.

 

2 25ml of ProteinA sepharose is in vast excess for 5ml of Ab, but this is the minimum amount that is easy to handle in a tube. If larger batches of protein are IPÕd the amount of ProteinA sepharose to be used should be deduced from the binding capacity of the beads (data sheet: 20mg of IgG/ml drained gel). The concentration of IgG in serum is 8-16mg/ml. 5ml of serum hence contains 40-80mg of IgG and 2-4ml of ProteinA sepharose (bed-volume) would be sufficient to IP all the antibody.