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immunoprecipitation concept - (May/13/2013 )

hello everybody,

i am sorry but i got a little confused with some concept of immunoprecipitation...

I pre-cleared my cell lysate with protein A beads.Then, i added antibody and incubate it for one hour with 35 ul of 50% bead slurry and followed by adding 1 mg of protein lysate ( from non denaturing buffer) . after washing and running the gel, i see a band from my input (30 ug) and was happy to see a band in my IP and no band in my negative control.

however, my questions are:

1. why i can not see the light chain and heavy chain? my target protein is around 50 which corresponds to the heavy chain, could it be the heavy chain? but my input corresponds to this band too.

2. how to really set a negative control? is it a protein that is known that does not interact with my target protein?

3. if my target protein binds to protein A in the presence of a ligand (group A) , but does not bind in the absence of a ligand (group B), if i do immunoprecipitation, can i still see this band in the group B? sorry i am confused on the concept, i might be misleading myself. i hope you can help me clarify this...

thank you...

-soymilk14-

you must be using antibodies from two different species. For example IP with antibody raised in rabbit, and western with primary antibody raised mouse. The secondary antibody during western is binding to your primary antibody in western, not IP.

We never pre-cleared with protein A. I'm not really sure if it's necessary in low volumes.

In IP you can incubate PBS with your beads as your negative control. This is clearly mentioned in SIGMA manual for protein A/G. I also did not throw away the supernatant after incubation. I used it directly in my western to see if the band of interest has disappeared from my lysate. It was usually +90% less.

-Curtis-

oh i see, thank you Curtis. yes, the IP antibody i used is from rabbit and the western primary antibody i used is raised in mouse. haha thank you. does that mean that my western signal of my target protein is not true IP? does that mean that i got false positive, that my target protein really does not bind (directly or indirectly) to my IP protein? sorry for this, my IP concept is not strong. i hope you can help me clarify this or suggest a good reading material for IP basic concept.

another thing is my third question:

3. if my target protein binds to protein A in the presence of a ligand (group A) , but does not bind in the absence of a ligand (group , if i do immunoprecipitation, can i still see this band in the group B? sorry i am confused on the concept, i might be misleading myself. i hope you can help me clarify this...


thank you so much

-soymilk14-

if you see a band, it means you have strong binding

-Curtis-

i see. thank you. i have another last question sorry... if i have two cell lysates: no treatment and treatment groups. Can i use IP to distingusih if my target protein can bind to my IP antibody in no treatment group and can not bind in the treatment group? is that the purpose of using non denaturing buffers for IP? sorry about this, i hope you can clarify for me. thank you

-soymilk14-