3 replies to this topic

[dllinpat]

sorry, I am a fresh man of biology, could anybody tell me what is the sensitive amount of protein for western blot?
I purified GST-fusion protein and checked by SDS-PAGE, bands were clear ,
but for western blot I think I have to dilute the GSTs , now I need to know the sensitive amount of it so that I can determine how many times the protein to be diluted.
thank you everybody, best regards to you!

[Prep!]

dllinpat, on Nov 2 2009, 03:55 PM, said:

sorry, I am a fresh man of biology, could anybody tell me what is the sensitive amount of protein for western blot?
I purified GST-fusion protein and checked by SDS-PAGE, bands were clear ,
but for western blot I think I have to dilute the GSTs , now I need to know the sensitive amount of it so that I can determine how many times the protein to be diluted.
thank you everybody, best regards to you!

it depends on the ptotein as well the the antibody titer value.
As a thumb rule, the western is atleast 10 to 20 times more sensitive than the SDS-PAGE (i am referring to the method to be used as a purity test.) If it is just for identity, 1000 times lesser amount can be loaded onto the WB. But also depends on the loading amount in SDS-PAGE
Good luck!!

[dllinpat]

thank you friend, I'm gonna try it now.

[Feelcontraire]

dllinpat, on Nov 2 2009, 02:25 AM, said:

sorry, I am a fresh man of biology, could anybody tell me what is the sensitive amount of protein for western blot?
I purified GST-fusion protein and checked by SDS-PAGE, bands were clear ,
but for western blot I think I have to dilute the GSTs , now I need to know the sensitive amount of it so that I can determine how many times the protein to be diluted.
thank you everybody, best regards to you!

You can choose to dilute your sample or the ab. There are some tables that compare sensitivities look at pierce supersignal.