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Possible Protein Aggregation Riddle


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#1 12358x

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Posted 08 April 2015 - 06:36 AM

Hi Everyone,

 

So this is my first time posting and I've got a bit of a riddle I'm trying to figure out, or at least make some sense of. I've run 3 blots, 1 as an antibody trial, and then 2 for actual data and while they both provide reasonable signal they have bands in different places. To make things more complex, a few conditions changed between the trial and the actual data blots but I can't figure out why I'm seeing the changes I'm seeing. 

 

For more information: I'm using a ferritin light chain (FTL) antibody and it should produce a band of ~20 kDa. In my trial using samples of Hippocampus, Cortex, and Spleen (as a marker) I produced a nice band at 20 kDa with a prominent minus primary band at about 35 kDa in the brain samples (not the spleen). When I ran my other two blots they were solely Cortex samples and the same Spleen marker. They were originally tested for a different protein and I stripped and reprobed for FTL. Now the samples show a prominent band above 220 kDa and the Spleen (same sample) shows a weaker band at 20 kDa and a prominent band above 220 kDa. The minus primary is gone but I've observed this multiple times after stripping a blot.

 

So the question is: Why have the bands moved? I know FTL can exist natively in an aggregate of up to 450 kDa but don't know what would cause the aggregation. The blots ran fine and the data from the initial antibody matched historical data perfectly. I can't imagine that the reprobe protocol would cause a shift (I would understand weaker signal though).

 

Any help is appreciated.

 

Extra Info: Using Criterion XT 12% gels with XT buffer and reducing agent (uses TCEP). The membranes are nitrocellulose. 

 

 

 

 

 

 

 



#2 bob1

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Posted 08 April 2015 - 12:34 PM

Try probing without stripping - in my experience, stripping causes more problems than it solves, as it usually nonspecifically attacks the proteins on the membrane and can cause loss of signal due to epitope removal.

 

The bands at 220 and 35 kDa are likely to be non-specific binding and as such should either be ignored or removed by careful titration of the antibody and wash steps. Remember that non-specific binding is not an indication that the antibody is working, and should not be used as an indication of the conditions for the blot being correct.



#3 12358x

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Posted 09 April 2015 - 07:37 AM

Hi Bob1, Thanks for the reply.

 

I'm fairly confident that the 35 kDa band is non-specific and due to my secondary antibody - I've previously identified it in control experiments. I'm a little curious about how the >220 kDa band would be due to nonspecific binding - I haven't seen it before in other experiments (stripped or otherwise) using the same secondary antibody. Is the suggestion that a reprobe protocol would sufficiently alter protein structure/binding to induce primary antibody binding?



#4 bob1

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Posted 10 April 2015 - 01:03 AM

I was meaning non-specific binding of the primary antibody for the 220 kDa band, which would then show up upon secondary detection.

 

I wasn't meaning that reprobing would alter the binding, I was meaning skip the stripping step if possible (if you are using HRP and different species primary ABs you can inhibit HRP with sodium azide so as to detect bands of similar size).






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