Dear everyone,
I am currently making a recombinant protein (protease) for checking its activity by zymography. However, before column purification (his-tag), the activity was shown only one band very clear.
But after purification, I check activity of this recombinant protein (by zymography) again, there are many bands smaller, also have activity but the M.M smaller than the predicted one. Does it mean my protein partially digested? Or something happened to my protein?
Please help me!
Thank you so much
Best regards,
Van
It's possible that the enzyme suffered from proteolysis during purification. This would result in bands with lower molecular weight.
If the protease is prone to self-proteolysis, purification without proteolysis may possibly be accomplished by performing the purification under conditions at which the proteolytic activity of the enzyme is very low, such as at a pH far from the optimum, in the presence of a non-covalent inhibitor (so that activity can be restored by dilution of the enzyme), or in the absence of an activating metal (for a metalloprotease).
it's possible that your protein underwent partial digestion or degradation during the purification process. Several factors could contribute to this...
To troubleshoot this issue, you may consider the following steps...
- Review your purification protocol to ensure that each step is optimized for maintaining the stability and integrity of your protein.
- Check for protease contamination by performing a control experiment where you purify a sample without the protease expression and assess its stability and activity.
- Consider using protease inhibitors during the purification process to minimize the degradation of your protein of interest.
- Verify the identity and purity of the purified protein using techniques such as SDS-PAGE and Western blotting.
- If possible, try alternative purification methods or conditions to see if they yield better results.
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