I am using CuBr/THPTA for a click reaction in total cell lysates. I am facing issues with my protein sample in non-reducing SDS-PAGE where it's not migrating properly and most of it remains at the top of the gel. Any suggestions for troubleshooting or alternative approaches?
Thanks.
PS: Can’t use BME due to experimental limitations.
Did you load another cell lysate or supernatant (not clicked) to confirm weather it is gel issue or with clicked sample ?
But, Sometime if sample is too viscous or sticky, very hard to load, you can dilute it with SDS running buffer or 1% SDS solution before loading.
The situation remains the same with non-clicked samples. Anyways, I will try again with dilute samples. I hope it works.
Thanks for your suggestion.
What percentage of the gel are you using? Generally, the higher the molecular weight, the lower the % of acrylamide used.
A higher % indicates that the pores formed will be smaller, making it suitable for smaller proteins, which will be able to easily pass through small pores. In short... you could be using the wrong % of acrylamide, so your protein won't be able to run very far through the gel and will be trapped at the beginning due to the "networks" formed.
For comparison: I work with a 112 kDa protein and use a 9% gel.
I hope I've helped, hugs!
https://www.abcam.com/en-gb/technical-resources/guides/western-blot-guide/electrophoresis
Hi! I am using 10% SDS-PAGE. But, It looks like there is a problem with my cell lysis protocol. Thanks for your suggestions.
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