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I am going to do an IP next week with your product ab69314. My plan is to process the acrylamide gel with comassie for excision of the bands and analysis by MALDI-TOF. Because of that I need as much protein as possible. I am planning to immunoprecipitate 3mg of my protein extract. How many ul should I add of ab69314 for that?. Datasheet recommends 20ul per IP but your example is performed with just 100ug and I don't know if with 3mg it is going to be the same. On the other hand can the elution be performed at 95ºC for 5 minutes? Can this elution be performed with the loading buffer sample 5x (Tris 60mM, glicerol 25%, SDS 2%, Bmercaptoethanol 14.4mM, Bromophenol Blue 0.1%) so it is ready to load?
Asked on Apr 23 2012
I recommend to test-run a smaller sample first. I can recommend to use 20ulslurry (2ug of IgG cross-linked beads)with 100ug protein and check the result. If this works optimal with your samples, I then can recommend to scale it up.
Since 3mg would mean 600ul slurry, I can recommend to optimise further. Maybe 250ul slurry will be enough (this is one vial of ab69314).
I am sorry I do not have a definitive answer to this question. This has not been experimentally tested by us and we therefore cannot recommend any definitive amounts.
I can confirm that elution of the protein after IP can be done by boiling in loading buffer. This will most probably also detach some of the IP antibody from the beads (althoughantibody and beads are covalently bound)and therefore there is the risk of getting heavy and light chains of the IP antibody into the gel.
Please check with your MALDI-TOF protocol for requirements on IP and gel run. .
Answered on Apr 23 2012