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Anti-his antibodies - Noob to protein work


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#1 moerae

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Posted 23 February 2012 - 07:43 PM

I've tagged my protein with His-tag and I'm using anti-his antibodies to probe it using Western blot. I'm overexpressing them in zebrafish and I've run a uninjected and injected samples and I'm seeing a band on my uninjected samples that's the same size as the band I'm also seeing it for my injected (overexpressing) samples. Could this be due to non-specific binding?

#2 mdfenko

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Posted 24 February 2012 - 12:56 PM

it could be. what is in your buffers to reduce non-specific binding? we use salt and tween in the washes and wash with blocking agent for the antibody solutions.
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#3 moerae

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Posted 26 February 2012 - 12:19 PM

I'm blocking with 5% skim milk powder in TBS-T, and washing with TBS-T. The primary antibody is in 2% milk powder with TBS-T and the secondary has 1% milk powder with TBS-T.

#4 mdfenko

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Posted 27 February 2012 - 08:43 AM

what is the size of the bands. there is an artifact that shows up around 60kDa (avg) that binds antibodies non-specifically.

you should also determine if the secondary binds there without using primary.
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#5 moerae

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Posted 28 February 2012 - 12:43 PM

At around the 50 KD mark. I'll check all those things you've mentioned.

#6 mdfenko

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Posted 29 February 2012 - 01:04 PM

if it is the artifact then it may be caused by keratins. they show up more and more as the reducing agent ages.

you can avoid it by using fresh reducing agent or, if possible, omitting reducing agent.
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#7 moerae

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Posted 05 March 2012 - 07:09 PM

Ok... I did as you suggested and tested out just the secondary antibody by itself... my whole blot lit up like a Christmas tree (a lot of non-specific bands).... I'm using the Supersignal Femto kit and found that I've been using more than the recommended amount of primary and secondary antiboies with it. If I reduce the antibody concentration would it fix my problem?

#8 mdfenko

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Posted 06 March 2012 - 12:36 PM

reducing secondary may help some but it is still binding, non-specifically, to your blot. if the whole blot lit up and not just in the area of the artifact then you may have to change your blocking agent and/or your secondary and, maybe, your primary. you may have a problem with the species in which the antibodies are made.

pictures may help
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#9 moerae

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Posted 06 March 2012 - 02:04 PM

I've stripped the membrane and trying to probe again with reduced primary and a new aliquote of secondary just to see if the secondary is contaminated. I've included a picture of the blot, sorry it's blurry, but you can see that the ladder lit up hugely but it had more than 10 bands which is surprising cos the ladder I used only had 10 coloured bands.

Attached Thumbnails

  • 2nd blot test 10 sec exposure standard.jpg


#10 mdfenko

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Posted 07 March 2012 - 09:06 AM

the ladder will have some traces of contaminating proteins (some may be unlabelled proteins from the set).

do you get the same or nearly the same pattern without the primary (using secondary only)? you can try adding normal serum from the species in which the secondary is developed to the diluent. you may also want to dilute more.
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#11 moerae

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Posted 07 March 2012 - 12:08 PM

I get nearly the same pattern without the primary. I'll see how I go with the diluted primary and secondary.




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