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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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Immunoprecipitation Equal Loading
Posted by: sockpuppettherapy (IP Hidden, New member, 1)
Date: January 16, 2008 10:04PM
Hi,
I'm currently doing experiments where we're looking to see interactions with and without treatment through co-IP. However, for several months I have been having a ton of difficulty trying to get equal loading of all four samples after the entire IP procedure. A gist of the protocol: 1. Lyse cells 2. Pre-clear samples with Protein G beads (200 microliters of bead slurry per sample) 3. Quantify protein concentrations after pre-clear and use equal amounts for co-IP - Use about 2 mg of protein in 750 microliters 4. Add antibody of interest and incubate overnight 5. Add 100 microliters of bead slurry and treat for 1 hour 6. Spin down and rinse beads in lysis buffer 3 times 10 minutes each 7. Add 100 microliters 2x SDS loading buffer and boil for 5 minutes 8. Load 40 microliters of each sample A couple of questions. 1. I've been spinning the beads at 12,000 RPM for 30 seconds, but have heard warnings about agarose beads "crashing" at these speeds (though the data sheet recommends this). Is this ok? I've tried lower RPMs but have had issues where the beads are not packed enough while I'm sucking off the supernatent. 2. Should I load all of the sample instead of putting in equal amounts? 3. I have been doing the western blot using chemiluminescence, which has also been a pain. Does anyone have any experience in alkaline phosphatase treatment, and does this tend to show more accurate readings of intensity? I know that I can also normalize the intensity and do a ratio, but I would very much like to get equal loading. Does anyone have any recommendations? Thank you.
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