purification of LDH was done from beef heart extract. my result were consistent with my classmates from the salting out part till the dialysis. but after i get my fractions from chromatography my LDH yield was much lesser than expected. wat could be the reason causing this? i need every possible reason to troubleshoot. thanks for helping.
2007-07-18 07:21:42 · 2 answers · asked by crystal 1 in Science & Mathematics ➔ Chemistry
Did you keep track of your enzyme activity in all of your fractions? If so, you should be able to determine whether your enzyme, for some reason, didn't bind well to the column, or whether your enzyme just because denatured. Did you loose a lot of total activity, or can you account for most of it, but just not where you wanted it to be?
So, one guess might be that your enzyme denatured and so you lost a lot of total activity. A second guess is that something in your sample prevented the LDH from binding to the affinity column, and so it just appeared in the unbound fraction rather than in the material that bound to the column.
A third possibility is that your enzyme was mis-handled after the chromatography, so that you lost activity after you had isolated it.
Those are the three easiest possibilities to think about.
2007-07-18 07:29:02 · answer #1 · answered by hcbiochem 7 · 1⤊ 0⤋
How about a few more details of your protocol?
2007-07-18 14:27:04 · answer #2 · answered by astazangasta 5 · 0⤊ 0⤋