Immunprecipitation with  Dynabeads

Modified from Invitrogen Protocol

 

Lysis Buffer (make up 10x solution and freeze)

Add the following immediately before use (final concentrations):

1 mM PMSF (serine protease inhibitor)

1 mM sodium orthovanadate (tyrosine phosphatase inhibitor)

1 Roche Complete Mini tablet per 10 mL buffer (protease inhibitor cocktail)

 

 

Low Salt Buffer

Components

For 500ml

50mM Tris pH 7.4

25ml 1M Tris pH 7.4

150mM NaCl

18.75ml 4M NaCl

0.2% TX-100

5mL 20% TX-100

2mM EDTA

10mL 100mM

2mM EGTA

10mL 100mM EGTA

0.1% SDS

2.5mL 20% SDS

 

 

High salt buffer

Components

For 500ml

50mM Tris pH 7.4

25ml 1M Tris pH 7.4

500mM NaCl

62.5mL 4M NaCl

0.2% TX-100

5mL 20% TX-100

2mM EDTA

10mL 100mM

2mM EGTA

10mL 100mM EGTA

0.1% SDS

2.5mL 20% SDS

 

 

 

Preparation of Dynabeads

1.     Completely resuspend Dynabeads by pipetting with a wide-bore tip or by rotating on a roller for 5 minutes.

2.     Transfer 25l beads per sample to a tube.

3.     Place the tube on the magnet to separate the beads from the solution, and remove the supernatant. Remove the tube from the magnet.

Binding of Antibody

4.     Add your antibody (1-10g per sample, typically 2g) diluated in 200l PBS w/ 0.02% Tween 20.

5.     Incubate with rotation for 1 hour at room temperature.

6.     Place the tube on the magnet and remove the supernatant.

Blocking

7.     Add 200l PBS w/ 0.02% Tween 20 and 1% BSA to the tube.

8.     Incubate with rotation for 30 minutes at room temperature.

Immunoprecipiation

9.     Pass lysates through a syringes 5x then spin down at 4C for 10m, 10 000x g.

10. Place the tubes with the beads onto the magnet and remove the supernatant.

11. Transfer supernatants to tubes with beads and resuspend the beads.

12. Place in 4C rotating end over end for 16 hours.

Washing

13. Take an aliquot of each wash buffer and add sodium orthovanadate to 1mM.

14. Wash the beads 3x in low salt buffer and 3x in high salt buffer. Keep on ice whenever possible.

15. Transfer the beads to a new, clean tube, place them on the magnet, and remove the supernatant.

16. Add 25l of 1x loading buffer with DTT.

17. Heat for 5 mins at 95C.

18. Place on ice to cool.

19. Put tube on magnet and load supernatant on gel for western blot.