I am working on a peptide that inhibits venom toxin activity. I wanted to know how the peptide is able to have inhibitory effects on venom proteins. To do that I need to figure out what proteins are being bound to the peptide. I have tried in-silico ways and got few hits but I would like to show it using some binding assays. Whats assays can I perform?
You could use a co-purification assay. Via addition of His-Tag N- or C-terminally to your peptide you could load it onto an Ni-NTA column or magnetic beads. Then you could incubate the column with your venom toxins mixtures, followed by wash steps to remove non-specific interacting proteins. Via elution of your peptide you would also elute interacting toxin proteins. Subsequently, these can be analyzed via SDS-PAGE followed by peptide-mass-fingerprint to clearly identify them.
Thank you, Sebastian Wenski! I guess His-tag cannot be added after we have purified the peptide. We have already mass-produced the peptide. Is there any other way to do it?
Siddharth Bhatia . You could try old fashion affinity chromatography by attaching your peptide to an activated resin and see what sticks to it.
There are many chemistries available depending on your peptide.
https://www.thermofisher.com/is/en/home/life-science/protein-biology/protein-purification-isolation/protein-purification/protein-immobilization.html
I don't know how complex your toxin samples are but you could also try to link your peptide covalently to the protein of interest with formaldehyd. Via a comparison of SDS-PAGE of a linked and non-linked sample you should be able to identify the your linked peptide-protein complex, cut it out, digest it and identify it via PMF.
Sebastian Wenski I am actually dealing with Viper venom which contains a cocktail of proteins ranging from 15-120KDa. If I understood it correctly, you are suggesting conjugating peptide to a protein to make it bigger and then check the interaction? There could be 2 issues here- 1) The peptide secondary structure might change that will impact the interaction 2) There could be additional binding pockets developed for the protein-toxin interaction that may provide additional binding partners that are not binding to peptide but to other pockets.
You can try the affinity chromatography approach. The peptide can be coupled to resin and then add the venom proteins. The bound proteins can be eluted and confirmed by mass spectrometry and other enzymatic assays. Fluorescence emission spectroscopy can also be used to show some interaction between the peptide and proteins. Hope this will help.
Thanks Archana Deka and Steingrimur Stefansson
I will try affinity chromatography approach initially. Hope it works :)
Archana Deka Can you elaborate on the use of Fluorescence emission spectroscopy for interaction?
@Siddharth if u atleast have some information for the bound proteins and can purify them..then u can use Fluorescence emission spectrometry..for example u can use different concentration of peptide and incubate with the purified protein and check the change in the emission spectra and compare with the spectra of individual components (peptide alone and protein alone).
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