Protocol Online logo
Top : Forum Archives: : General Lab Techniques

Glycerol for enzyme buffer - (Aug/19/2006 )

I have to prepare enzyme buffer in lab and its reference book is 50 percent glycerol is added.
I just want to confirm that i in my lab prepared enzyme buffer i have to put a 50 percent glycerol.
I am not adding glycerol and useing it with out glycerol. what you say.
regards

-samita-

Most commercially obtained restriction enzymes are supplied in a 50% glycerol solution.

-karyotyper-

Usually enzymes are stored at -20°C in 50% glycerol to avoid freezing of the enzyme.
Then you have to dilute at least 10 times your enzyme because you should not have more than 5% of glycerol in your reaction buffer.
If you freeze your enzyme without glycerol, you can kill its activity, especially if you repeat several time freezing/thawing.

-Missele-

hi,
in general glycerol stabilizes the protein, as well protects hydrophobic sites in the protein.

regards
payeli.

QUOTE (samita @ Aug 19 2006, 11:59 PM)
I have to prepare enzyme buffer in lab and its reference book is 50 percent glycerol is added.
I just want to confirm that i in my lab prepared enzyme buffer i have to put a 50 percent glycerol.
I am not adding glycerol and useing it with out glycerol. what you say.
regards

-payeli-

I have enterokinase enzyme form Neb compay and I am dialyizing my protein against its specific buffer with out glycerol. I have enzyme as 2 ug/ml and volume is about 100 ml.
How much this enzyme to dilute for tag removal.
2 ug/ml means .002 ug/ ul.
Its a just a ordinary confirmation experiment because i donot know about the exact concentation of my protein. If some body have an expericen using enterokinase and its dilution then please suggest me
I am diluting it for 10 times and using 2 ul in 20ul reaction for tag removal.
guide me
regards

-samita-