Immunoprecipitation

Immunoprecipitation Protocol by Adam Kimbrough from Laboratory of Dr. Debi Fadool

1. prepare lysis buffer on ice a. 750ul ilysis buffer into homogenizer and 750ul added to wash out after homogenization

b. add 100 ul PMSF, 10 ul P, L, A protein phosphatase inhibitors per 10ml of lysis buffer

2. harvest fresh tissue

3. homogenize tissue 50 strokes on ice in 750 ul lysis buffer with PPIs until completely homogenized

add 750 ul lysis buffer to wash the homogenized tissue

use a siliconized pipette to transfer homogenized tissue to tubes

4. rotate in rotary for 30 min at 4 degrees Celsius to allow to lyse

5. Centrifuge for 10 minutes at 14000 rpm (setting #14 on fadool centrifuge) at 4 degrees Celsius

6. Remove supernatant to fresh tube and save pellet in case it contains protein

7. Fraction supernatant

Take 50 ul of supernatant per IP antibody for a lysate fraction from each sample

Take 20 ul per antibody for Bradford assay from each sample

Take remainder for IP pre-clear split into tubes for equal amounts per IP antibody

8. Pre-clear with protein A sephirose beads Reconstitute sephirose into a homogenious mixture by raking against ependorf tube rack and shaking

Cut pipette tip for sucking up of sephirose

Add 30 ul sephirose A protein beads to each sample (i.e. 30 ul in HDAC and 30 ul in 14-3-3 tube)

Rotary for 1 hour minimum (can be longer) at 4 degrees Celsius

9. Run Bradford assay and calculate amount for equal protein per sample into the IP (fractions should have same levels of protein but samples from different animals shouldn’t) Calculate sample protein levels for volume to add for IP

10. Centrifuge for 10 minutes at 14000 rpm at 4 degrees Celsius

11. Save supernatant from pre-clear into new tube (beads[pellet at bottom] collected the antigens present to discard)

12. IP overnight

Add appropriate amount of pre-clear soluation to tubes for equal amounts of protein as calculated by Bradford assay (see step 9)

7.5 ul neat antibody (for HDAC and 14-3-3, calculate for others) for ~750 ul of IP fraction per sample

rotary at 4 degrees Celsius overnight with antibody in each tube

finish IP the following day

13. Add protein A sephirose beads for 3 hours Reconstitute as above

Rotary in 4 degrees Celsius for minimum 3 hours to collect IP product to beads

14. Centrifuge for 10 minutes at 14000 rpm (NOW SAVING PELLET-has IP product) Siphon off supernatant and rinse with Wash buffer

15. Repeat centrifugation for 10 minutes and wash with wash buffer 3 times (can leave small amount of liquid)

16. Final rinse remove all liquid and leave only pellet

Add 30 ul of KFL to each sample and store at -20 degrees Celsius as long as needed

17. Samples are now ready for running on Western Blots

use 15 ul of each sample per run (2 runs/sample per IP)