Digest Buffer Precipitation - Na2HPO4, L-Cysteine, EDTA (Jul/17/2008 )
I'm having all kinds of problems with this buffer. It is a standard buffer used with Papain for cartilage digestion. Here's the recipe:
5 mM L-cysteine
100 mM Na2HPO4 dibasic, anhydrous, molecular biology grade
5 mM EDTA
All in ddH2O
pH: 7.5
For digestion: Add 500 micrograms/mL papain, heat to 70 C for 4 hours, cool quickly.
This buffer tends to precipitate quickly from the heating/cooling process, and also precipitates in approximately 2 weeks if left at room temperature (25 C). The pH also skyrockets during the precipitation, rising to well over 8 and 8.5. This seems to be the exact opposite effect I desire from a buffer.
Upon further testing, the papain has nothing to do with the precipitation, as it occurs from just the stock buffer solution if put through the same process.
Has anyone else had this problem? Or am I just making an elementary mistake? We are using this buffer for GAG assays, and the precipitation and pH increase necessitate immediate assay once the buffer is created. I would much rather have a stock of this on hand, instead of having a time-intensive assay requiring preparation of a buffer before each run.
Thanks for any help!
5 mM L-cysteine
100 mM Na2HPO4 dibasic, anhydrous, molecular biology grade
5 mM EDTA
All in ddH2O
pH: 7.5
For digestion: Add 500 micrograms/mL papain, heat to 70 C for 4 hours, cool quickly.
This buffer tends to precipitate quickly from the heating/cooling process, and also precipitates in approximately 2 weeks if left at room temperature (25 C). The pH also skyrockets during the precipitation, rising to well over 8 and 8.5. This seems to be the exact opposite effect I desire from a buffer.
Upon further testing, the papain has nothing to do with the precipitation, as it occurs from just the stock buffer solution if put through the same process.
Has anyone else had this problem? Or am I just making an elementary mistake? We are using this buffer for GAG assays, and the precipitation and pH increase necessitate immediate assay once the buffer is created. I would much rather have a stock of this on hand, instead of having a time-intensive assay requiring preparation of a buffer before each run.
Thanks for any help!
No idea, but see if you can find your solution in these links. Let us know what was the trouble, if you do find out.
http://search.vadlo.com/b/q?p=1&sn=158...rel=0&srt=0
..
I'm currently running through a few tests to isolate the problem. I'll report back when I've discovered the culprit.
5 mM L-cysteine
100 mM Na2HPO4 dibasic, anhydrous, molecular biology grade
5 mM EDTA
All in ddH2O
pH: 7.5
For digestion: Add 500 micrograms/mL papain, heat to 70 C for 4 hours, cool quickly.
This buffer tends to precipitate quickly from the heating/cooling process, and also precipitates in approximately 2 weeks if left at room temperature (25 C). The pH also skyrockets during the precipitation, rising to well over 8 and 8.5. This seems to be the exact opposite effect I desire from a buffer.
Upon further testing, the papain has nothing to do with the precipitation, as it occurs from just the stock buffer solution if put through the same process.
Has anyone else had this problem? Or am I just making an elementary mistake? We are using this buffer for GAG assays, and the precipitation and pH increase necessitate immediate assay once the buffer is created. I would much rather have a stock of this on hand, instead of having a time-intensive assay requiring preparation of a buffer before each run.
Thanks for any help!
No idea, but see if you can find your solution in these links. Let us know what was the trouble, if you do find out.
http://search.vadlo.com/b/q?p=1&sn=158...rel=0&srt=0
..
I can only guess but I assume that the phosphate cause the problem and precipitates over the time; try to use both EGTA and EDTA to be sure to reduce free calcium (impurities from cysteine?)
After further inspection, the only combination which is causing a problem is Phosphate and Cysteine. I made solutions of all 3 combinations of chemicals, heated them to 70 C for 4 hours and rapidly cooled on ice (as I would do with a digest). After centrifuging the samples, only the combination of Phosphate/Cysteine had any precipitate in it. Phosphate/EDTA and Cysteine/EDTA showed no traces of precipitate. I don't think it is a purity issue, as the Cysteine/EDTA solution had no precipitate. I'm currently looking through the literature to see if I can find a possible interaction between Cysteine and Phosphates. I'll keep you informed of progress.
Edit: Looking closer - the Cysteine/EDTA solution DID have some precipitate, so it appears that the Cysteine is the problem. It could be that there are calcium impurities as you have suggested. I may try purchasing newer, more pure cysteine. Or, I may try using a combination of EDTA/EGTA as you have suggested. Thanks for the tip, and I'll keep you informed.