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Secondary Antibody reacting with Loading Buffer? Is this possible?


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#1 torontostudent

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Posted 14 January 2010 - 11:10 AM

I have been having problems with my Western blots.
Basically, I am getting bands developing in the wrong lanes.

I have tried to optimize my Western protocol to get a single band. So I have done the required controls including:

- lysate of interest
- lysate from tissue that does not contain antigen
- primary + secondary
- secondary only
- loading buffer blank (with no lysate from any source)

I am getting the strangest result. I am getting bands in my loading buffer blank. They are high molecular weight, 3 bands, always in the same spots. No matter if the primary or secondary is there.

I have done the same blocking as recommended (5% skim milk for 1 hour). Lots of washes.

I even made a gel and ran only blank loading buffer and probed with secondary and still got the same bands.

Has anyone seen this before?

What do I do?

I am obviously now going to try a different secondary but I do wonder what it could possibly be reacting with in just the loading buffer.

#2 mdfenko

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Posted 14 January 2010 - 12:17 PM

how high molecular weight?

if they are in the range of bsa then you may be seeing an artifact thought to be caused by keratins from dust. it appears stronger as the reducing agent (especially 2-mercaptoethanol) ages.

Edited by mdfenko, 14 January 2010 - 12:17 PM.

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#3 torontostudent

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Posted 14 January 2010 - 12:56 PM

they are in the range of 75-100 kDa.

#4 mdfenko

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Posted 15 January 2010 - 08:14 AM

do these bands also appear in the samples?

your loading buffer may be contaminated.

or your water may be contaminated (did you use water to dilute the loading buffer in your blanks?).
talent does what it can
genius does what it must
i do what i get paid to do




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