i have expressed and purified my protein according to the Ni-NTA protocol and successfully got 1 sharp band size around 40kDA which is my protein size.
But why each time i do western blot it did not detect anything except control protein?
i've been doing this many times using the crude lysate and also purified protein, but there were still no detection on the film.
can somebody help me please..
i juz confused what band was actually being purified?but it was the right size..

Hey Kema,

Not to worry we'll figure things out. First what are your controls to insure the antibodies are working correctly?

Rb

kema said:

i have expressed and purified my protein according to the Ni-NTA protocol and successfully got 1 sharp band size around 40kDA which is my protein size. But why each time i do western blot it did not detect anything except control protein? i've been doing this many times using the crude lysate and also purified protein, but there were still no detection on the film. can somebody help me please.. i juz confused what band was actually being purified?but it was the right size..

i'm using recombinant protein from seaweed as a controlled.my antibodies (anti-his hrp conjugates) was ok, because it can detect the control protein.
is it because my protein was not cencentrate enough to be detected compared to the control, or what?

Kema sorry for the belated reply, had to take off to Brazil to run some experiments. My guess is the band you are seeing is not your protein. It doesn't make sense especially if you're controls are all working and you can see a band on an SDS-PAGE gel after Ni-NTA purification that it isnt concentrated enough. Let me know how its going. Again apologies for the tardy reply.

Rb