I'm doing immunoprecipitation for the first time. For the pilot experiment, my aim was to pull down ABCA1 using protein A/G agarose beads and then probe for phospho forms. I used 2 ug per sample of an antibody that we are familiar with for Westerns and is labeled for IP.
I ran a gel with the IP samples, the lysates that were the source material and the protein A/G agarose beads. After transfer, I stained with Ponceau S and saw the following:
IP samples: no bands
Lysates: some bands, mostly lower than the expected size
Beads: no bands
I started with 250 ug of protein, which I understand may not be enough. Additionally, the protein A/G agarose beads were from 2011 (opened vial, stored at +4). Further, the samples were spun at about 4000 gs instead of the recommended 2000 gs.My question is, which is most likely to be the cause of the apparent failure- insufficient protein to start, old beads, or beads crushed by excess cetrifigal force? Or another issue...Thanks much. Below is the basic protocol:
- Prepare samples (frozen tumor lysates): 250 ug total protein in 250 uL lysis buffer with 2x inhibitors
- Add 2 ug ABCA1 antibody to samples. Incubate at +4 overnight, rotating
- Next day: Add 40 ul protein A/G agarose beads, incubate at +4 rotating, for 4 hours.
- Spin at 2000g cold centrifuge 2 minutes**. Save lysates (supernatant) for gel as control.
- Wash with 500 ul cold PBS 3 x.
- Add 5-10 ul buffer w/2x inhibitors to beads. Add 5 ul of 5x reducing sample buffer; mix gently.
- Boil 10 minutes; place on ice.
- Spin briefly to precipitate beads.
- Collect supernatant. Add lysis buffer and loading buffer to beads.
- Run SDS PAGE gel at 150 volts, 3.5 hr, +4.
- Wet transfer overnight, 30 v +4.
**Actually g force turned out ot be higher, about 4000, because of the centrifuge used. Some sources say this can damage the beads, but I'm not sure...
Edited by Artemis2007, 01 August 2014 - 06:46 AM.