Hi.

I'm planning to perform ELISA to find amino acid residues which directly bind to the protein X (protein of interest).

You may see the design of the experiment in the picture that I posted.

In the experiment, His-tagged protein X (affinity purified) is bound to Ni-coated 96 well plate (Thermo), and would be incubated with each biotinylated synthetic peptides 1, 2, 3 and 4. Among these peptides, the peptide having the highest affinity will be decided by using the TMB substrate.

Preparing the experiment, I met up with some questions for which I need help from someone has expertise.

1. What would be the range of appropriate amount (or concentration) of the His-protein X and biotinylated peptides to use?

2. What can I use as a negative control for the peptides?

3. What buffer should I use? Commonly used buffers for ELISA such as PBS+1%BSA and PBS+0.05%Tween for blocking and washing, respectively? Or may I use the binding and washing buffer below, which are prepared for in vitro protein binding assay?

- Binding buffer : 50mM Tris (pH 7.5) + 150mM NaCl + 2mM EDTA + 1mM Dithiothreitol + 0.1% NP-40 + 5mg/ml BSA

- Wash buffer : 50mM HEPES (pH 7.5) + 150mM NaCl + 1mM EDTA + 1mM DTT + 0.1% Tween-20

I appreciate for all the answers and wish you luck on every work.

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