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purpose of input in IP


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#1 soymilk14

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Posted 17 September 2009 - 03:00 AM

hello everyone,

i am new in learning immunoprecipitation. i want to know what is the purpose of the input? i transfected the cells with a certain protein x and wants to determine if this protein can pull down stat5.

i collected the input and loaded it as follows:

1. cell without transfection
2. cell transfected with the protein

i collected also the supernatant mixed with sephadex g-beads as follows:

1. cell without transfection
2. cell transfected with protein

I also detected it first with antibody against actin, the protein x and stat 5.

I wanted to know what should my expected results be like? And what will show up in my input? And what is the purpose of the input?

thank you, i hope you can give me a link on how to read more on immunoprecipitation. thank you....

#2 laurequillo

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Posted 25 September 2009 - 01:58 AM

Your input shows first that you have the same amount of stat5 in both lines. And second it shows that you dont have the protein x in the first line, and when you use the antibody antiX (the tag you were using) you did not precipitate stat5, so the precipitation of stat5 is due to the presence of you protein x
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