Immunoprecipitation for MS
Equilibrate the bead slurry to room temperature.
Take 100 ul beads and add 0.4 ml Binding/Wash Buffer to the tube and invert with gentle
shaking. Centrifuge tube and remove supernatant, keep beads. Repeat this wash step 3-4
times.
To the beads, add 250 ul of binding/wash buffer and transfer to a new microcentrifuge
tube. Apply 1-10 μl of antibody prepared in 0.3-0.4 ml of Binding/Wash Buffer.
Cap tube, rock/rotate for at least 15 minutes to allow the antibody to bind to the gel.
Centrifuge the tube and keep the beads.
To beads add 0.5 ml of Binding/Wash Buffer and transfer to another microcentrifuge
tube. Invert tube 5-10 times. Centrifuge tube and discard flow-through. Repeat this step
two additional times using the same collection tube.
Add 250 ul binding/wash buffer to beads, transfer to new tube. Beads are ready for the
IP.
Binding/wash buffer: 140 M NaCl, 8 mM sodium phosphate, 2 mM potassium phosphate
and 10 mM KCl, pH 7.4
For the immunoprecipations, we have used ~250 ul of sample (either postnuclear cardiac
lysates, or purified microsomes depending on the situation). For our proteomic studies,
we have used the postnuclear lysates.
We dilute the sample 1:1 with Binding/Wash Buffer
Rotate for at least 16 hr (overnight) at 4°C.
Wash with binding/wash buffer 4-5X (with rotation; 5 min), keeping beads
Elute with 100 μl of elution buffer (0.1M glycine, pH 2.5), rotate 10 min, spin keep
supernatant, repeat with 90 ul elution buffer (total of 190 ul elution)
To elution, add 10 ul of 1M Tris HCl ph 7.6-8.5 to bring the final pH of the solution up to
~7.4-7.6.
We then take this eluate and digest it directly in solution with trypsin, solid-phase
extracted and then run on a mini-MuDPIT (4 hrs separation) compared to the usual 21
hour for the complex ventricular fractions.