I'm using purified recombinant proteins to coat for Elisa. As a negative control, I used only the vector without the insert. Reactivity is seen also in that vector. How to troubleshoot this?
You have to avoid non-specific binding, this could arise from not properly blocking the surface or the methodology that you are using.
Your antibody may be recognizing a protein in the parental cells as well as the intended antigen. You may be able to remove this reactivity be pre-adsorbing the antibody with an extract from the parental cells.
Thank You Mr. Omar and Mr. Adam.
Mr.Omar, I'm using 5% Skim milk in PBST as blocking buffer.
Mr. Adam, my primary and secondary is of same origin. Still do I have to pre-adsorb the antibody?
The secondary antibody should not be from the same species as the primary antibody. It should be raised in a different species against the IgG from the species in which the primary antibody was raised. The primary is usually raised in mouse or rabbit. The secondary is usually goat anti-mouse or goat anti-rabbit, although there are certainly many other possibilities.
It is unlikely that the secondary antibody will cause background, but you can test for that by leaving out the primary antibody.
Just to clarify. You say you are using purified recombinant proteins. So your control preparation from vector without insert should not have any protein in it - is this correct?
If so, only the blocking protein is present in the control wells no? If not, which protein is coming from your insert free vector?
In any case, you have some form of non-specific binding, either from your primary or your secondary. You can do a variety of tests.
1. Use wells that have no protein (i.e. purified recombinant or vector control). Block sets with different types of blocker - BSA, skimmed milk, NAP etc. Then run the assay in one set with primary and secondary and in another set with secondary only. You'll get a good idea of where the nsb is coming from.
2. Check whether your vector only preparation has any proteins in it. Carry out a western blot on this with the primary and secondary that you use for ELISA and include a positive control lane with your recombinant protein.
When you say that your primary and secondary are from the same origin, do you mean same supplier? or same species? It is difficult to understand how they can be from the same species unless the poor animal has some severe form of autoimmunity to it's own antibodies - in which case one would assume it wouldn't survive long enough to be useful.
Thank You Mr. Adam and Mr. Deepan for your valuable suggestions. I got a clear idea regarding the secondary antibody. I'm so sorry that I mislead by saying as its of same origin. Unknowingly, I meant the species only. Yes, I agree that it can never be of same origin.
I have attached the SDS PAGE gel pic of the recombinant proteins. Kindly give your inputs. Thank You.
Hi. So what is the protein in your "no insert" control?
Is your desired protein a fusion with something on the vector?
You can easily test whether your primary or secondary are binding to that band in the vector only control using a Western blot.
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