Ethanol and DMSO for FITC preparation - (Aug/23/2007 )
Hello, you scientists ! I got a problem on FITC preparation . Because I'd like to add peptide-FITC mixture into bacteria and animal cell. And according to Sigma's protocol , FITC is soluble in anhydrous DMSO(5 mg/ml) or in ethanol(20 mg/ml) , but considering my peptide was solved in ddH2O, DMSO added will produce heat. And ethanol may cause cell toxicity.
If I'm going to use Ethanol , what the concerntration I should prepare for to prevent cell toxicity? Or if another solvent i could choose ?(protocol also says acetone, water, 2-methoxyethanol may be solvent too) Or DMSO ?
I also think abuot using water as solvent, but FITC decomposes in water and my experiment has a long time duration(about 6 hours). What should I do ?
Any advices plz , ~
Is your peptide heat sensitive? You can add FITC solution in small portions over a period of time. DMSO is better than EtOH. The reaction is usually quick, so you may not need 6 hr to complete, 30 min to 1 hr is OK. Be sure to use pH control during reaction and not to use Tris as buffer. NaHCO3 is fine.
Yes NaHCO3 1M as a solvent for your peptide, then you just need to add a very little amount of FITC dissolved in DMSO, so the solvent is not a problem. And, anyway, you should purify by size exclusion to get rid of the unreacted FITC, that way, in the end, you will have your peptide dissolved only in the solvent you choose for elution.