Chloroform Extraction of NAD/H - (Jan/22/2007 )
I need to do a chloroform extraction of yeast cell extracts to isolate NAD/H. I currently add an approriate (double) volume of chloroform to a lysis mixture (which contains broken cells, potassium phosphate, and ammonium acetate). I usually let the mixture separate on ice for 10 minutes (while I tend to another part of the protocol). I then remove the aqeous phase, using a pipettor, for analysis.
I need to extract as much NAD/H as possible so that I can get an attamole/cell reading and I'm afraid I'm losing sample during the extraction. Are there any techniques available to help out?
This depends on the method you use to detect NAD/H. What happens if you don't do the extraction step? Unless there is an awful lot of lipid I don't think you'll get lipaemic interference at 340nm if you use spectroscopy. It's a long time since I worked with yeast but breaking up the chitin layer may be the hard part. How are you lysing the cells? Lastly, would you get better yield without the ice?Ice for proteins and peptides for sure but maybe its not needed here.
We're quantifying the NAD/H using spectroscopy on an HPLC. The cells are being lysed using bead and a bead beater which seems to be efficient. I hadn't thought about skipping the ice. I'm worried that active proteins may convert NAD and NADH thus messing with the total cellular concentrations of each. Do you think that would be a problem?
Skipping the extraction step is a thought. I definitely don't want to clog the HPLC column, but I'm sure I can remove larger proteins without a problem, right?
Thanks for your help!
Do you use a guard column on your chromatography?
Yes, we do. I suppose it would be worth trying to spin out some of the larger proteins, doing a chloroform extraction on them (to remove bound NAD/H), adding the extract to the cell lysate, and putting that through the column. I'll give it a go. Is there anything you can think of that I should be careful about? I'm very new to HPLC and don't want to overload or ruin the system.
Thanks so very much for your help!