Hello, my name is David
I want to study the inhibitory effect of the pAbs in my protein. So I incubated the pAbs with my protein in solution. But when I analysed the complex formation by gel filtration I only detected the two particles separately. The same pAbs can recognised the protein when we do a Wb or ELISA. Does anyone know how to explain what is happening and/or can provide a solution?
Thanks!
David
I'm not sure what you mean by detecting the particles separately. Where you following the protein peaks off the gel filtration by UV absorbance? What resin were you using? One possible reason for not forming an immune complex in solution, but seeing bands on Western blot or detecting by ELISA might be the reactions are being performed at different pH or different ionic strength - yes/no? I learned immunology many years ago, and know that immune complexes form best at antigen:antibody equivalence. At antibody or antigen excess, binding is univalent and immune complexes don't form. I hope this helps.
Dear Dr. David Vizarraga
The intriguing scenario unfolds as your trusty pAbs throw a party in Western blot and ELISA, but surprisingly, they're playing hard to get in the gel filtration dance. They're whispering secrets in one setting and giving the cold shoulder in another. What's the deal? Well, let's dive into the molecular gossip:
Epitope Masquerade:
Complex Tango:
Buffer Compatibility Drama:
Concentration High Jinks:
Technique Mix-Up:
Control Experiments Showdown:
and so on.
AB Bayazid
It has certainly been observed many times that some antisera are more effective against denatured protein (such as western blots or ELISA) and others against native protein. This is due to the nature of the primary epitopes the pABs react against. There are certainly many other possible reasons and A.B Bayazid gave a good list of them
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