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Fusion tagged protein and Western Blot problem


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#1 Biochem_newbie

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Posted 11 April 2011 - 10:01 AM

Hi there

I have a Myc tagged protein. When I first received the plasmid, transfected, etc, I was able to see the Myc tagged protein band during Western, but now when I run it, I am not able to see the band. When I blot with the antibody raised against my specific protein, I am able to pick up the band with no issue.

Is there any reason why the myc Ab won't detect my myc tagged protein?
I thought I was diluting it too much but even with a small amount, I still can't get anything.

Any comment's would be greatly appreciated!

Thank you

#2 bob1

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Posted 11 April 2011 - 03:47 PM

Antibody has gone off? Do you have another protein that is also myc tagged that you can use to check whether it is the antibody or the protein?

#3 Biochem_newbie

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Posted 12 April 2011 - 05:07 AM

There is a possibility that during this time I have been working with my protein, the myc tag might have broken off?

#4 bob1

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Posted 12 April 2011 - 03:32 PM

Not necessarily broken, but perhaps your plasmid is degraded by freeze/thaw or DNAse action. It could also be that the plasmid has been mutated by the bacteria while you were growing it up.




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