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High background with FLAG antibody


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#1 MaggieRoara

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Neutral

Posted 02 March 2009 - 01:26 AM

I had initially tried detecting using my ATP1B1 primary antibody followed by antimouse HRP secondary antibody, but this gave no signal. I then did a dot blot using FLAG primary antibody and antimyc HRP secondary antibody on the same samples. The dot blot did have lots of background for FLAG but i could see the "dot". Dot blot for the beta actin Ab somehow didnt show up. I applied the same conditions for the initial blot after stripping. I have nice bands for my control beta actin primary Ab, antimouse secondary Ab. But for the FLAG Ab, i see a big blob in the shape of my blot.
help please


confused

Edited by MaggieRoara, 02 March 2009 - 01:27 AM.


#2 bob1

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Excellent

Posted 03 March 2009 - 03:18 PM

try changing your blocking conditions, and/or shortening the time you have your secondary incubating for.




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