IP for beginners? - (Jun/01/2007 )

hi everybody,

i have to start a whole series of IP experiments in the next weeks and i was wondering what exactly is the input control people show in publications or on seminars. is it the supernatant after spinning down the beads (after the beads bound to your IP-Ab) to check for equal protein concentration? sorry, i know it is really a greenhorns question, but i was really wondering.

regards,

Lab Dance

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in order to estimate the amount of immunopreciptated protein? it may work but protein determination must be done very carefully several times with at least two independent methods;

you have to wash the immunoprecipiates and will lose some unspecific binding proteins; you also may monitor the amount of protein in your wash buffer

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Normally the input is what you use for IP, that is the lysate before the first Ab is added. So input is the starting material, what you originally have before IP

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okay. thank you very much.
By the way, is there actually a formula for preparing a 50% Prot. A solution?? i realized that all my colleagues rather estimate the amount of beads they dissolve. i think this is not very nice ad i am unsure how much to use. espacially as these beads are too cheap. maybe someone has an answer. thanks already in advance.

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By the way, is there actually a formula for preparing a 50% Prot. A solution?? i realized that all my colleagues rather estimate the amount of beads they dissolve. i think this is not very nice ad i am unsure how much to use. espacially as these beads are too cheap. maybe someone has an answer. thanks already in advance.

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