Friday 5 November 2010

GGS LIVE - Immuno and co-immunoprecipitation

Whatsup you all?

GGS LIVE sections presents today an immunoprecipitation method:)

Method: Immunoprecipitation.

About: Protein precipitation using specific antibodies is a technique that allows for enrichment of a particular protein and components associated with this protein (it is a type of protein purification).

What: Immunoprecipitation using anti-myc antibody from DT40 cells stably expressing 3myc tagged protein.

This technique can be used to purify the protein of interest in order to use it in a specific assay or to identify its potential interacting partners. For example an enzyme protein kinase can be immunoprecipitated and subsequently used in the kinase assay. The cartoon below show immunoprecipitation steps.

Briefly, cell lysate is prepared by cell lysis in IP buffer of choice (the composition of the buffer might be different from protein to protein analysed and may have to be found empirically). Cell lysate is also sonicated to share the DNA that may interfere with the immunoprecipitation process and also to solubilize proteins. Next, cell membrane and debri is spun down and protein concentration is determined. On this stage the lysate is ready to go. We also should prepare our beads and antibody. The Protein A or G beads are washed three times with lysis buffer (aka IP buffer) to remove beads storage solution (usually ethanol which can interfere with the precedure). Washed beads are mixed with antibodies. After coupling, beads are mixed with previously prepared cell lysate and this is our immunoprecipitation step. After precipitation beads + antibodies + protein of interest + partner proteins are washed with lysis buffer (or other buffers) to remove unspecifically bound material. Finally, precipitated proteins are eluted and analysed by Western blotting (for the Western blotting tutorial visit this post GGS LIVE - Western Blotting). Ok lets see the results now...

On the top panel we see bands representing the protein against which the antibody was used (immunoprecipitated protein). Bottom panel shows co-precipitated protein (a partner protein of the myc-tagged protein of interest). The lanes are: input lane, representing starting material, unbound lane showing material that left after the immunoprecipitation and finally the IP lane which represents the precipitated proteins fraction. The control IP has to be performed so we are sure that the protein we are enriching for is specifically pulled down. One can imagine that protein of interest might bind to other part of the antibody (that is common for anti-myc or control antibody) or bind unspecifcally for example to the beads. Simply saying the signal form our protein should be present only in the experiment IP but not in the control IP. As you can see this is correct for our myc tagged protein which is present in the starting material, it is depleted in unbound fraction and there is an obvious enrichment of it on the beads (IP lane). In the case of the control IP again it is present in the input lane but it is absent in the IP lane (which is perfect:). 
The same samples were analysed for the presence of a partner protein that is known to interact with our myc-tagged target. You can clearly see that the interacting partner is also present in all the samples beside the control IP what suggests a that it is interacting with the myc-tagged protein. Of course novel interactions have to be confirmed with reciprocal experiment.

I hope u enjoyed and understand that immunoprecipitation is a very powerful cell biology technique:)



1 comment:

  1. Hi Maciek,
    I would be interested to use your IP-cartoon. Could you tell me where you got it from? Or did you do it yourself? (I would need a reference...)
    Thank you very much!
    BTW - nice blog and good explanations!
    Best, Anne