I performed crosslinking using a 10 mg pellet and 5 ml of diluted 2.5% glutaraldehyde for a period of 2 hours. Upon attempting to dissolve the crosslinked enzyme for activity assay, I encountered inaccurate dissolution. Moreover, when compared to the free enzyme, the crosslinked enzyme did not exhibit activity in the activity assay. However, increasing the concentration of the crosslinked enzyme resulted in activity, which is in contrast to the behavior of the free enzyme, which exhibited activity at a concentration of only 100 ng. What further steps can I take to address this issue?
The activity of the cross linked material is likely being lost because of excessive modification of the enzyme molecules, resulting in distortion of the structure. I would withdraw samples over a time course and check both activity and degree of cross linking by SDS PAGE. You need to find the point at which the reaction should be stopped. Using a lower concentration of glutaraldehyde will give you more control and more time to do any in process tests. It is worth noting however that glutaraldehyde cross linking will never give a defined end product. Why do you wish to cross link the enzyme? There are potentially better ways of doing it.
Nick Gee Hi sir,I understand your point, but it's my advisor's decision, and I can't change it to another approach. However, my advisor now realizes that this approach won't work for my enzyme, so I need to find a new, more cost-effective approach for immobilization. If you know of any suitable and more affordable approaches, it would be greatly beneficial for me.
Cyanogen bromide conjugation possibly would be a good choice for immobilizion while preserving the enzyme activity...a type of mild biologic attachement procedure....
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Peptides