I´m having some trouble with my Western Blot experiments (SDS page). I would like to know what are the steps to standardize a reaction.
I tested one sample with beta actin antibody and it shows 2 differents bands (one of them is the correct size).
Then I tested mchr1 antibody and couldn´t see anything.
I also noticed dirt on the membrane when revealed.
What specific reaction are you referring to?
If you see two bands in a Western blot with an actin antibody, and one is the right MW then the other could be a degradation (lower MW) of actin or if it is higher MW than actin, you could have a dimer or oligomer.
By MCHR1 are you referring to melanin concentrating hormone? If so, then you have two different proteins you are looking at. Does the assay/reaction use both?
By reaction, do you mean the Western blot probing with antibody and final reaction of peroxidase with substrate or the general procedure?
I need more details to figure out what is going wrong and why you have dirt? or is it a non-specific coloring of the blot?
What specific reaction are you referring to?
If you see two bands in a Western blot with an actin antibody, and one is the right MW then the other could be a degradation (lower MW) of actin or if it is higher MW than actin, you could have a dimer or oligomer.
By MCHR1 are you referring to melanin concentrating hormone? If so, then you have two different proteins you are looking at. Does the assay/reaction use both?
By reaction, do you mean the Western blot probing with antibody and final reaction of peroxidase with substrate or the general procedure?
I need more details to figure out what is going wrong and why you have dirt? or is it a non-specific coloring of the blot?
I agree with Gustavo, it is hard to render an opinion without seeing the blot.
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