Hey,
I performed several acetone precipitations with eluates (1ml) of my IP´s in 50 mM Tris buffer. I usally dissolve the pellets in 1xSDS buffer and load a SDS-gel. Now I had to switch to a HEPES buffered system. The IP workes fine and the western blot looks similar to the old experiments - but after the precipitation of the HEPES buffered sample I get a very big almoust unsoluble pellet (I tryed Urea, SDS, sonification, heat), which I was´nt able to load on a gel. Is it possible that the HEPES salt is not optimal for aceton precipitation followed by sds PAGE?
Is the HEPES salt maybe also precipitated and the Tris is not?
Thank you very much in advance
JEtte
Hey Jette,
your Problem sounds like not optimal conditions. I always used the protocol of sigmal-aldrich to prepare my samples with IP for SDS-Gel and afterwards blotting.
Here is the Link - so you are able to compare with your protocol.
Greeting
Christian
http://www.sigmaaldrich.com/life-science/cell-biology/antibodies/antibodies-application/protocols/immunoprecipitation.html
Thanks christian, I think I did´nt make my point clear enough:
My IP performance:
-Incubation of the lysate on beads (anti HA)
-washing the beads (3-5 times)
-elution with HA peptide (peptides diluted in lysis buffer) ~ 1ml
-acetone precipitation of the HA eluate
disolve the protein pellet in sds/lämmli buffer ~25µl
load the gel
The IP workes fine but the point is , that I changed the complete buffer system (for cell lysis, washing, elution) from Tris buffer to HEPES buffer.
Lot of greetings
Jette
What are the other components of your HEPES buffer? Potassium and SDS form an insoluble precipitate.
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