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.
The negative control for the peptide could be the same peptide without the biotin. Another negative control could be imidazole, to compete with the His tag for binding to the nickel surface.
A single well of a 96-well ELISA plate has a limited binding capacity. The maximum amount of protein per well is probably less that 1 microgram. The capacity in terms of moles of protein will likely depend on the size of the protein.
The binding affinity of the peptide for the protein is going to be a key factor, If the affinity is high, then you can get as much as 1 peptide molecule bound per protein molecule. If the affinity is too low, the peptide will just wash away and you will get no signal.
Hi,
If you are performing this experiment for the first time, in order to decide the concentration of protein or peptide to be used in the experiment, you have perform concentration gradient for both protein(0 uM- 10uM) and peptide (0 uM - 100 uM) in two different experiments. With this you will come to know the optimum concentration that works for both protein as well as peptide.
With respect to the buffer system, I have used the PBS + 3%BSA for blocking and PBST for washing in the similar experiment and it worked pretty well.
Adam B Shapiro
Deekshi Angira
Thanks Adam and Deekshi for helpful suggestions!
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