WangLab:Immunoprecipitation and Immunoblotting

Day 1

 * 1) Prepare lysis buffer and keep it in ice
 * 2) *Lysis Buffer (Non-strigent example):
 * 3) 3X wash cells with ice-cold PBS
 * 4) lyse the cells with lysis buffer, 200 l for each 10 cm2 area confluent cells, scrape the cell debris using the blunt end of the pipette tips, collect the cell lysis and kept in ice.
 * 5) centrifuge top speed in 4oC micro-centrifuger for 10 min
 * 6) transfer supernatant to new tubes
 * 7) measure the protein concentration
 * 1:4 mixture of protein reagent: water
 * 1) *Take out 5 ul out from 1 ug/ul BSA and cell lysis samples, read the absorbance by photospectrometer and calculate the protein concentration of lysis samples.
 * 2) Take out 400 ug of protein from each sample, add in 2 ug of antibody, rock for 1 hr in 4oC.
 * 3) add 30 ul of protein A or protein A/G beads to each sample (note: gentle tap and resuspend all the beads), Rock for overnight at 4oC.

Day 2

 * 1) Prepare resolving Gel (typical 10%), takes 30 min to solidify.
 * 2) Prepare Stacking Gel (mostly 5%), add in comb to generate wells. Take 5-10 min to solidify.
 * 3) centrifuge the beads for 5 min at 3000 rpm at 4oC. aspirate the supernatant and add in 1ml lysis buffer for each sample.
 * 4) repeat step 9 for 2 times, centrifuge the beads for 5 min at 3000 rpm at 4oC. aspirate the supernatant.
 * 5) add in 6X SDS protein loading buffer to each sample