These peptides are not on resin anymore and i do have Wang resin to maybe conjugate them back on the resin before attaching the dye but i am not quite sure how to do so!
Is there a better way to do this?
I am in agreement with Guobing Xiang.
Instead of HPLC, I have had success purifying labeled peptides using a long column of Sephadex LH-20, eluted under gravity with distilled water. After collecting the fractions containing the labeled peptide, the water can be removed by lyophilization. Warning: FAM is damaged by too much exposure to light, so keep it in dim light or darkness whenever possible. You will need some way of determining which fractions from the columns are the ones you want.
add 5(6)-Fam NHS ester into peptide in buffer@~pH 8.3 under shaking or stirring.
Why resin? Your peptide will not come with resin, your peptide has terminal NH2 at least!
Yes that is correct. Since all the labelling is always done on a resin i presumed the peptide has to be on a resin.
The NHS ester will react with the unprotonated alpha-amino group and the unprotonated epsilon-amino group of any lysine residue in the peptide in solution. The solution should not contain any other competing amines. A 0.1 M sodium bicarbonate solution is often used for water-soluble peptides. The excess ester must afterwards be removed by some form of chromatography, such as reverse-phase HPLC.
Hanieh
Unless you asked a special order for your peptide on resin, otherwise your peptide will come without resin. Follow Adam's suggestion, 0.1M NaHCO3 is the easiest way, pH will be aroud ~7.25-7.3.
Adam B Shapiro
Thank you for your answer.
Can i use PBS instead of sodium bicarbonate?
Is the HPLC step necessary?
Hanieh
Yes for sure, you can use PBS buffer, make sure the pH is 8.25 to 8.5. HPLC purification is the best way, since peptide is not big molecule, you can not use dialysis. You might be able to use Sephadex column or so。
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