Cell Culture Lysate Preparation and Immunoprecipitation for Dynal® Dynabeads®
 
Required Materials:  

Dynal® Dynabeads® (species specificity depends on the origin of the immunoprecipitating antibody)

Dynal® Magnetic Particle Concentrator (MPC)

1.5 ml microcentrifuge tubes

15 ml conical tube

End-over-end rotator

Tabletop microcentrifuge

Required Solutions:

 

Phosphate Buffer Saline (PBS) pH 7.4
 8.06 mM Na2HPO4-7H2O
 1.47 mM KH2PO4
137.9 mM NaCl
 2.67 mM KCl

Lysis Buffer
 30 mM Tris-HCl pH 7.5
150 mM NaCl
 20 mM Mg-Acetate
 1% CHAPS or Igepal-CA-630

0.2 M triethanolamine pH 8.2
Stock triethanolamine is 7.4 M (1:37 dilution)
To make 3.7 ml: add 100 µl of 7.4 M triethanolamine to 3.6 ml of ddH2O

50 mM Tris-HCl pH 7.5
To make 10 ml: add 500 µl of 1 M Tris-HCl pH 7.5 to 9.5 ml of ddH2O

Tris-Buffered Saline (TBS)
 20 mM Tris-HCl pH 7.5
500 mM NaCl

XL Buffer
 20 mM DMP (dimetyl pimelimidate dihydrochloride)-[5.4 mg/ml]
200 mM triethanolamine pH 8.2

IP Buffer
 30 mM Tris-HCl pH 7.5
150 mM NaCl
 20 mM Mg-Acetate

Sample Buffer
62.5 mM Tris-HCl pH 6.8
   2 mM β-mercapto-ethanol (β-ME)†
  10 %  glycerol
   2 %  Sodium Dodecyl Sulfate (SDS)
0.01 %  Bromophenol Blue

† Dithiothreitol (DTT) can be used instead of b-mercapto-ethanol
‡ Pyronin Y can be used instead of Bromophenol Blue, or the buffer can be clear

Protocol:

Preparing the Dynabeads®:

1) Wash 100µl of species-specific Dynabeads® (depending on the origin of your precipitating antibody) three times with 500 µl of 1X PBS by repeated pelleting using the Dynal® Magnetic Particle Concentrator (MPC)

2) Add 5 µg of antibody in 500 µl of 1X PBS following the final wash.

3) Incubate from 1 hour at 25°C to overnight at 4°C while rocking end-over-end in order to bind the antibody to the beads.

4) Pellet beads with the MPC and remove the supernatant.

5) Wash the beads twice for five minutes with 500 µl of 1X PBS at 4°C, rocking end-over-end during each wash. Pellet the beads with the MPC between washes.

6) Pellet the beads with the MPC and remove the last wash.

7) Re-suspend the beads in 500 µl of 0.2 M triethanolamine pH 8.2 and incubate for 10 minutes at room temperature while rocking end-over-end.

8) Pellet the beads with the MPC and remove the 0.2 M triethanolamine.

9) Re-suspend the beads in 500 µl of XL Buffer and incubate for 30 minutes at room temperature while rocking end-over-end.

10) Pellet the beads with the MPC and remove the XL Buffer.

11) Add 500 µl of 50 mM Tris-HCl pH 7.5 (room temperature) and incubate for 15 minutes while rocking end-over-end in order to stop the cross-linking reaction.

12) Pellet the beads with the MPC and remove the 50 mM Tris-HCl pH 7.5.

13) Wash the beads three times for five minutes with 500 µl of 1X TBS, rocking end-over-end during each wash.

14) Store the beads by rocking end-over-end at 4°C in 500 µl of 1X TBS until lysate is prepared.

Preparing the Lysate:

1) Grow cells on one to four 10 cm tissue culture plates.

2) Wash the plates twice with 10 ml of 4°C 1X PBS.

3) Scrape cells from each plate into 1 ml of 4°C 1X PBS and transfer to a 15 ml conical tube.

4) Pellet the cells by centrifugation at 2000 x g for 2 minutes at 4°C.

5) Re-suspend the cell pellet in 1 ml of IP Buffer and transfer to a pre-weighed 1.5 ml microcentrifuge tube.

6) Pellet the cells by centrifugation at 2000 x g for 2 minutes at 4°C.

7) Remove the supernatant and re-weigh the microcentrifuge tube.

8) Re-suspend the cell pellet in an equal volume (w/v) of Lysis Buffer (i.e. a 500 µg pellet should be re-suspended in 500 µl of Lysis Buffer).

9) Lyse the cells by rocking end-over-end for 30 minutes at 4°C.

10) Spin the cell lysate in a microcentrifuge at 17,000 x g for 15 minutes at 4°C in order to remove cellular debris.

11) Transfer the supernatant to a clean 1.5 ml microcentrifuge tube and store at 4°C until ready for pre-clearing.

12) Solublize the remaining pellet in a volume of 1X Sample Buffer equal to the volume of the supernatant that was removed in the previous step.

Pre-clearing:

1) Wash 100 µl of Dynabeads twice with 500 µl of 1X IP Buffer using the MPC (use the same species-specific beads as are used for the actual immunoprecipitation).

2) Remove all of the final wash from the beads and add the recovered lysate from above

3) Incubate the lysate and empty beads by rocking end-over-end for 2 hours at 4°C.

4) Pellet the beads with the MPC.

5) Remove and save the cleared lysate for immunoprecipitation.

Immunoprecipitation:

1) Pellet the antibody-bound beads prepared earlier with the MPC and remove the 1X TBS.

2) Add the cleared lysate prepared above to the antibody-bound beads.

3) Incubate the lysate and antibody-bound beads by rocking end-over-end for 2 hours at 4°C.

4) Pellet the beads with the MPC. Remove and save the lysate.

5) Wash the beads four times with 1ml of IP Buffer at 4°C, rocking end-over-end during each wash. Pelleting the beads with the MPC between washes.

6) Solublize the beads in 30 µl of 1.5X Sample Buffer.

7) Heat the beads in Sample Buffer for 10 minutes at 70°C.

8) Pellet the beads with the MPC. Remove and save the supernatant.

9) The sample is now ready to be analyzed by SDS PAGE.

 

© 2008 Joseph T.E. Roland
www.Cytographica.com