I'm looking for a protocol to fluorescently label blood platelet factor 4 with Rhodamine. The standard protein labelling protocol doesn't seem to work. Any ideas from anyone?
Hi,
did you make labelling in alkaline conditions (carbonate-bicarbonate pH9.2)?
Yes, I did the labelling at pH 9 in a carbonate-bicarbonate solution
Hi,
which standard protocol do you mean? Are you using an activated ester for your labeling? Which solvent did you use for the rhodamine?
Hi Sandra,
I am using an activated NHS activated ester in attempting to label the amine group of the protein.
By 'Standard protocol' I mean the protocol recommended by the dye manufacturer. i.e.
1 part Sodium bicarbonate adusted to pH 9 added to 20 parts PBS buffer to give a labelling buffer of pH 8.3.
Rhodamine is dissolved as 1 mg in 100 ml of DMSO,
Protein is dissolved in the labelling buffer (1 mg/ml)
The Rhodamine and dissolved protein are then mixed to give the required degree of labelling and later purified using a Sephadex G-25 column.
I have a feeling that the positive charges of the amine and rhodamine are inhibiting the labelling.
hi Richard,
please note that Rhodamine is a trivial name for a class of dyes. Please tell us (Answerers) what a kind of rhodamine (p.e. dye with 2 carboxyl groups/ What a kind of isomer a.s.o. ) Are you using TAMRA , Rhod Red or Rhod green with NHS ester?
JRG
Hi Richard,
did you use fresh DMSO, which is free of water? Do you have the possibilty to do an pilot experiment with your dissolved dye and a short lysine-containing peptide or another nucleophile e.g. TRIS or something like that to be sure, that your activated ester is still an ester after dissolving? Maybe you have the possibility to do LC-MS analysis...
Best regards,
Sandra
Jean Rene: The precise dye i am using is ATTo-550 NHS ester which according to the product datasheet uses a rhodamine derivative and is stated as being similar to TAMRA/CY3.
Sandra: Yes I used fresh amine free DMSO. Regarding the short pilot experiment, I will have to check if we a short lysine containing peptide readily available in our centre.
Is it essential to use rhodamine as a label? If a blue-fluorescent label would be acceptable, too, give fluorescamine a try. Fluorescamine reacts with primary amines, works under mild conditions, is easy to use, and yields results in seconds. As with all fluorescent tags, you have to verify that your protein still functions normally.
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