Another way to do this is to use fluorescence polarization. The peptide is synthesized with a fluorescent label (fluorescein or TAMRA, for example) attached at a position that is unlikely to be involved in binding. The fluorescence polarization of a nanomolar concentration of the peptide is measured as the protein concentration is increased. If the peptide binds, the fluorescence polarization will probably increase. A graph of polarization versus protein concentration can be used to measure the dissociation constant. A control experiment is to then compete the labeled peptide off the protein using the unlabeled peptide.

Another way to do this is to use fluorescence polarization. The peptide is synthesized with a fluorescent label (fluorescein or TAMRA, for example) attached at a position that is unlikely to be involved in binding. The fluorescence polarization of a nanomolar concentration of the peptide is measured as the protein concentration is increased. If the peptide binds, the fluorescence polarization will probably increase. A graph of polarization versus protein concentration can be used to measure the dissociation constant. A control experiment is to then compete the labeled peptide off the protein using the unlabeled peptide.

Take in consideration the size of magnetic beads, protein and peptide because to avoid steric hindrance. Instead, you may immobilize your protein on a solid support (like titre plate) and use a fluorescent-labeled peptide for studying the interaction. After a washing step, you can measure the fluorescence.

Thank you very much Dr. Shapiro for your most valuable suggestions as usual. I will try this. However, the facility is not available so I will push it little towards pulldown assay first. I wanted to confirm from you that, suppose the no. of moles of peptide is way higher than that beads, is there a possibility that it wouldn't bind at all to the beads. Also the protein concentration is very high in the reaction mixture. And Buffer is also one issue I am thinking. Are these factors affect the binding a lot ?

Mahmoud El-awady Thank you for your suggestions. So what should be the ideal situation in general for better binding ? I usually elute it by boiling the sample (bead+ Protein+ peptide) together and run the gel. I find no binding, most of the protein I can see in the unbound fraction. :(

Boiling? is this method included in the manufacturer instructions? you mentioned that you are using an Invitrogen product.

I think boiling may result in dissociation of the peptide from protein. Try washing with PBS (pH 7.4).

Mix the biotinylated peptide with the streptavidin beads. Remove any excess unbound peptide from the beads because it will compete with the beads for binding to the protein. This is easily done by settling the beads, removing the supernatant, adding buffer, and repeating a few more times.

Then add the protein to the beads. Allow some time for binding, keeping the suspension well-mixed. As a control, use beads with no added peptide. Let the beads settle. Remove the supernatant. Wash the beads with a suitable buffer in which you think the interaction between the protein and the peptide is stable. Do not use a large volume for the wash because the high dilution could cause the protein to dissociate from the peptide. Instead, do about 3 small-volume washes.

If the protein is bound to the beads at this point (based on SDS-PAGE of the beads), you can try to elute it by adding the peptide that is not biotinylated. You can probably even get a measure of the affinity of the interaction this way by varying the concentration of unbiotinylated peptide.

The positive control is to elute with the peptide that is biotinylated to compete the peptide off the streptavidin as well as to compete the protein off the beads. This should tell you the total elutable protein amount.

The choice of buffer for this experiment could be critical. The interaction may be highly sensitive to pH and salt concentration. Non-specific interactions between the peptide and the beads, or between the protein and the beads, could occur. You may be able to reduce this by adding a bit of nonionic detergent such as Tween-20, or by adding a bit of salt.

The peptides interaction can be obtained mathematically by performing the surface or volume integral for both interaction molecules or physically by using the Lennard_Jones potential law. You must do some calculations for physical paramaters, such as surface or volume atomic densities, attractive and repulsive constants, and the radii

, surface area and volume for each proposed structure.

For “weak” protein-protein interactions — from roughly mid-micromolar to low-millimolar — protein-observed solution-NMR spectroscopy (protein NMR), biolayer interferometry (BLI) and surface plasmon resonance (SPR) are used most often.

For stronger protein-protein interactions, low-micromolar to nanomolar, isothermal titration calorimetry (ITC), fluorescence polarization (FP) are commonly employed, as well as BLI and SPR. (ITC and FP each can measure into mid-micromolar range, say 50 uM.

https://www.malvernpanalytical.com/en/products/technology/microcalorimetry/isothermal-titration-calorimetry

https://www.ncbi.nlm.nih.gov/pubmed/25859974

studying protein-peptide interactions is not trivial, you'll definitely need to find the best magnetic bead from Dynabead options, eg M-280 Streptavidin or MyOne Streptavidin T1, and optimize all conditions. But should be doable, there are papers reporting successful application of this methodology

Hi,

Protein-protein interactions are often very short lived and weak interactions - Very interesting work was also done by the group of Albert et al. 2007 (Nature 450, 683-694) who studied nuclear pore complexes and interactions followed by downstream mass spec.

Albert et al used the Dynabeads™ M-270 Epoxy in combination with optimized buffers as these beads have very low background to avoid contaminate the preparation. They did spend a significant amount of time to find the correct buffer composition to capture these weak interactions.

These buffers are part of Dynabeads™ Co-IP kit which includes Dynabeads™ M-270 Epoxy and Ab coupling buffers in addition to co-IP buffers offering the opportunity to fine tune weak and transient interactions between short amino acid sequences in different proteins in terms of stringency.

feel free to get in touch at [email protected]

kind regards

ketil