Hydrophobic peptide (53%), 15 a.a.-long contains a single Cys-residue and Amine free, can be dissolved in DMF. I need to attach Alexa Fluor647-maleimide to the thiol group of the peptide and Alexa Fluor 647-NHS ester to primary amine.
How can I get doubly labeled peptide?
I dissolved dyes in DMSO and i used 5 mM TCEP for disulfide bounds. also I added 1% SDS in the reaction.
you can if your AF materials are good. In the last couple years, in my experience, Life Sciences (who bought Invitrogen who made the original AF dyes) have not been providing good material.
When Invitrogen first made the dyes they had their scientist make a huge stock pile of dyes and then they fired most of them. That stock pile started to run out out in 2010. I have not had any luck to get good coupling from any of the new stock.
What you have written in your question is the proper way to do the coupling though so if your material good then you should be able to get dual labeling.
Good luck
The best way is to do it a lot of times using a cheap dye for optimization (Fluorescein-NHS or Fluorescein-Maleimide). The optimal pH, target, and dye conjugate concentrations are crucial for getting efficient and specific coupling of the NHS ester dye. For the thiol, you must use a good reducing agent (old TCEP is no good... use freshly made DTT if possible), and ideally work under anaerobic conditions or aerobically but very quickly with a bit of EDTA to minimize thiol reoxidation before dye addition to your sample. Also if you have large amounts of dye you can just add an excess of dye to the reduced sample containing the DTT but that's a waste of dye if you ask me. Good luck!
Thanks Christopher and kambiz,
I have a positively charged peptide (IP:8.25), so i think i have a problem by non-specific dye-dye or peptide-dye interactions. I tried with Alexa647M(-charge) and Atto647N M (+charge) and Atto655 (neutral). I did not get any result by different dye, buffer, pH, incubation time and .....
but I can label the peptide with NHS ester one at least.
So, I should try with EDTA.
If your peptide is big enough to separate from DTT using a spin column you should reduce with 1mM DTT then remove the DTT and exchange into buffer with 1mM EDTA with the desalting spin column and immediately add the dye for the labeling. The pH of the thiol labeling reaction should be about 7.5. Also you will need about 5-10 fold excess of the dye. Best
Isn't it possible for you to do the N-capping with the active ester before cleavage/deprotection? Alexa should be good with the TFA standard cleavage cocktails.
I think you are mentioning in the peptide synthesis process but i ordered the peptide from company and i am not familiar with that process.
Yes, I meant during the synthesis.
A good general reference for chemical labeling is the book by G. T. Hermanson "Bioconjugate Techniques". You'll find detailed protocols and conditions there for almost everything.
http://amzn.to/1c9lwd9