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

  1. Prepare lysis buffer on ice
    • 750ul ilysis buffer into homogenizer and 750ul added to wash out after homogenization
    • 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)