In ELISA, the conformation of the target protein is often important. If you lyophilizate your protein, you might place your protein in water to reduce the concentration of salt. This might disrupt the 3D conformation. If you keep the protein in the buffer, the lyophilisation will extract water but not salt and again the 3D conformation will be lost due to high salt concentration. In western blot, this 3D conformation is less important.

You might try to concentrate your protein using a centricon or any device like this one. It will reduce denaturation.

Hi Prabhu,

Stabilizers and preservatives that are commonly being used in Lyophilization may sometimes interfere in binding of proteins in ELISA system. Sometimes Lyophilization also causes conformational change in protein structure or may damage the epitope regions on the proteins.

You can dialyze or desalt the lyophilized protein and try to test it in ELISA.

Good Luck.

Hi all,

thanks for the replies. @ Shiv, should I dissolved the lyophilized protein in some buffer and again dialyzed against some buffer to get back the original conformation?

Hi Prabhu,

You can only remove stabilizers or salts from your lyophilized protein by dialysis/desalting but you cannot restore the original conformation of the protein once it is altered/damaged due to heat stress during lyophilization process. However i recommend you to desalt the lyophilized protein using centricon (molecular weight cut-off low binding centrifugal filters) since it is fast and reduce the time as happens with lengthy dialysis. Just try this process by dissolving the protein in water for injection or distilled water.

Good Luck.

This may be a silly suggestion, but have you tried setting up the elisa with the same primary antibody you are using for the western blot? Quite often elisa test kits make use of similar, but not exaclty the same primary antibodies.

Hello, My another doubt towards the same is, if the 3D structure is disturbed by lyo then how come researchers use this technique to produce large quantities of protein for NMR spec and X ray for structure determination?

When you run the protein in your WB is it in a reduced state? it could be that your antibody actually only reacts with unfolded protein, where the tertiary confirmation is disrupted (i.e. if the antigen is a peptide sequence). Another reason for failure of your ELISAs is if the protein is not sticking to your plate. Did you confirm attachment?

I confirmed that the positive control where the bait protein coated directly onto the plate probed with the antibodies gave very good results though the same protein coated over any cytokines or matrix proteins gave very poor binding results.