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Anti-Penta His antibody detecting extra bands

western blot

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#1 Mafer

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Posted 20 September 2016 - 04:54 PM

Hi,

 

I'm using anti-PentaHis mouse antibody and Goat anti-Mouse secondary HRP antibody, to detect a heterologously expressed 6His tagged protein in HEK 293T. The things is, there is always an extra band near 60kD, which even appeared in  negative control, so I'm sure it is not my protein. May I know if anyone met this problem before and is there anywhere that things could have gone wrong and I missed out?

 

Thanks a lot in advance.



#2 mdfenko

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Posted 21 September 2016 - 03:23 AM

we would need more information to be certain but, you may be seeing an artifact caused by keratins from dust and mercaptoethanol.

 

have you tried a blank sample (no protein)?


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#3 Mafer

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Posted 12 March 2017 - 12:45 AM

It's been some time and I've found out why. :D

The band is also in blank cells - but not blank wells.

There was a publication regarding this. So, it was not seen in any publication using His Tag because most of them cut out that part.

 

Here is the paper that sort of answered my question:

https://www.ncbi.nlm...les/PMC4459774/







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