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Antibody against H1N1 cannot recognize epitopes

western blot plant virus

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6 replies to this topic

#1 Curtis

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Posted 18 April 2014 - 12:28 AM

We have produced a series of plant viruses that have epitopes from proteins of H1N1. The epitopes have different sizes but none are more than 40 aa. All of the epitopes have been added to the c-terminal of a plant virus protein, so they don't have their own start or stop codons, obviously. The problem is that our western blot with antibody against H1N1 cannot label anything on the membrane, maybe because it doesn't recognize the epitopes.

 

Computational analysis show that the epitopes must be outside the surface, however I don't think it would make a difference since we are doing wb.

 

We have not added any whole gene of H1N1 into our plasmid. Do you think it is more likely to be detected by the antibody if a bigger portion of the protein is present? Suggestions will be appreciated.



#2 bob1

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Posted 18 April 2014 - 12:48 AM

My understanding of antibodies raised against full length proteins is that the antibody doesn't actually recognize the full length protein, something like 80% of the antibody complement will be made up of antibodies that all recognize the same short epitope, which is usually about 4-8 amino-acids long.  Perhaps your tags don't contain this region?



#3 Curtis

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Posted 18 April 2014 - 02:28 AM

Each one has a different length. Im not sure what's wrong. The funny thing is that the mouse-raised antibody against H1N1 binds to wt plant virus on the membrane. Im really puzzled.

#4 bob1

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Posted 19 April 2014 - 12:54 AM

Non-specific binding I would guess, it's well known for antibodies to do this occasionally, especially if you are using a non-conventional (i.e. not mammalian/bacterial/yeast) system such as your plant viruses.

 

You could try a peptide competition to see if the wt binding goes away or not.



#5 Curtis

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Posted 20 April 2014 - 03:36 AM

the reason we want to detect the chimeric virus is that we actually want to purify and separate it from the wt. Our system generates both. the final molecular weight is very close and both viruses give bands only few kDa apart. not sure how to separate them if anti-H1N1 can't bind to the epitopes.



#6 bob1

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Posted 21 April 2014 - 06:12 PM

Yes, that would be a problem.  Would it be possible to do different tags and separate them by affinity purification?



#7 Curtis

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Posted 22 April 2014 - 02:44 AM

That's actually what I suggested and we're studying it.





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