WangLab:Immunoprecipitation and Immunoblotting
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Immunoprecipitation and Immunoblotting:
- Prepare lysis buffer and keep it in ice
- Lysis Buffer (Non-strigent example):
|2M Tris/Hcl (ph7.5)
|100 mM Na3VO4
|100 mM PMSF
|10 mg/ml Leupeptin
- 3X wash cells with ice-cold PBS
- 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.
- centrifuge top speed in 4oC micro-centrifuger for 10 min
- transfer supernatant to new tubes
- measure the protein concentration
- 1:4 mixture of protein reagent: water
- 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.
- Take out 400 ug of protein from each sample, add in 2 ug of antibody, rock for 1 hr in 4oC.
- 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.
- Prepare resolving Gel (typical 10%), takes 30 min to solidify.
- Prepare Stacking Gel (mostly 5%), add in comb to generate wells. Take 5-10 min to solidify.
- centrifuge the beads for 5 min at 3000 rpm at 4oC. aspirate the supernatant and add in 1ml lysis buffer for each sample.
- repeat step 9 for 2 times, centrifuge the beads for 5 min at 3000 rpm at 4oC. aspirate the supernatant.
- add in 6X SDS protein loading buffer to each sample
|SDS Gel-loading buffer:
|Tris.Cl (PH 6.8) (mM)
|Dithiothreltol (DTT, mM)