I have Heparanase III and Chondroitinase ABC which I would like to use to cleave the heparan sulfate and chondroitin sulfate chains off their core proteins before I run the samples on a western blot so I can visualise the core protein bands clearly (without the GAG-associated smear).
I have seen this done in many papers but there doesn't seem to be a specific protocol for when/how much/in what conditions/for how long the samples are treated with these enzymes. Is it best to treat the cell pellet before or after lysing them? Is there anything in a typical lysis buffer I should avoid? Or maybe it is better to treat the membrane itself after the proteins have run? How long/what concentration/what conditions should I use and how might I go about optimising that?
So many questions! Any protocols, papers with info or advice would be greatly appreciated!
Stjepan,
Thank you for your in-depth response, this has given me insight into a few important variables I hadn't considered. I appreciate you explaining the "peeling effect" in alkali, I have been researching this but was having a difficult time extrapolating it for my purposes.
I will expand on these ideas to come up with a protocol to test. If all goes well I will report back with results!
Thank you again.
Best regards,
Tayler
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