What is Far Western Screening - (May/30/2006 )
Does any one happen to know what is " far Western screening". One more question, what is "Kozak's rule"?
(I am a traditional biology student..... )
Thanks in advance.
western-blot is detection of a protein on a nitrocellulose membrane or PVDF membrane by an antibody coupled to an enzyme, or by an antibody recognized by a second antibody linked to an enzyme.
far western-blot is used to detect protein protein interaction. instead to use an antibody to detect the blotted protein, you use a protein (that is not an antibody).
the Kozak rule says that the translation starts at the first AUG downstream the transcription start.
for far western you are suspecting a protein protein interaction. Ex A interacts with B but you need to confirm really.
So in first, you run a full protein extract on a SDS-PAGE gel. Transfer it. You first blot with your antibodies A and B.
Then you strip the membrane, and submit it to a renaturation process. That means the proteins on it will be quite folded as normal cellular conditions.
You label for ex B (35S methionine or a DIG labelled aminoacid) and incubate your membrane with labelled protein.
Normally, the signal from B protein should overlap the signal obtained in western blot.
For final confirmation, same assay should be done with A labeled protein, and that time, the signa overlap the signal obtained with antiB antibody.
Thank you, Missele and fred_33.
I understand most part now.....