Hello All,
I have prepared a protein marker using proteins expressed by my lab. Now, I am interested in making a pre-stained marker and I need guidance for how to make a marker pre-stained using dyes i.e. Coomassie blue
I assume that you want the markers for separation (electrophoresis, ultracentrifugation...). CBB is unsuitable for this, as it does not bind covalently to the protein and would be lost during the separation process. You can use either amine-reactive probes like FITC, or SH-reactive probes like IAEDANS. Because both the isothiocyanate and the iodacetamido-group can bear different coloured dyes, you can, if desired, label all your proteins in different colours. Part III of my "Biophysical chemistry of proteins" (978-1-4419-7250-7) deals with protein modification.
Thanks you Mr. Buxnaum for your guidance, I also have done skimming of part in your book you mentioned. It was really helpful in developing concepts but the thing is I am using resources already in lab i.e. dyes (G-250, R-250 etc.) as I am bound to utilize them instead of ordering new materials.
If you may guide in this regards, I will be obliged. I am thinking of making 1% mix of suitable dye in Glacial A.A and adding it to my ladder followed by heat treatment.
Hi Ali Faiq The dyes will have to be covalently attached to the proteins to be useful as SDS-PAGE markers.
If any of the dyes you in your lab have free -NH2 or -COOH groups, you can do simple EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) coupling.
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