Hi all,
I'm working on bacteriocine! My proteins don't want to seperate, i m trying 120V for 4 hours, and 7.5% is the concentration of my concentration gel. Do you have any suggestions for me to regulate that, Thank you.
Which molecular weight range of proteins are you expecting?
Might be you need to change the gel % from 7.5% to 16%
Article Purification and Characterization of a Novel Bacteriocin Pro...
When you say your proteins don't want to seperate, are all the samples running as a single coalesced line ?
Hi Soumaya,
As Bhairavi says, it is necessary to know the molecular weight of your protein. I also find surprising you need 4 hours for the SDS-PAGE. May you give us some more details to understand the problem and try to help you?
Thanks for your reply, the molecular weight of my proteins is around 90KDa. I have noticed that Even after 4h migration the proteins bands are still stuck in the concentration gel and don't make it to the seperation gel.
If you use laemmli sample buffer or using similar composition, you should observe bromophenol blue dye at the bottom of the gel after PAGE run. Be sure about your sample buffer and preparation protocol for its denaturing capability. At this stage, I recommend you to examine an alternative protein sample and re-test the platform for the PAGE process. Then take into consideration the current transmission problems and working electrode malfunction. Even protein ladders can validate the system. You should prepare the fresh SDS running buffer and see the bubbles when you apply the voltage if everything works properly. As an alternative, you can test precast gels in place of handcastings (if you use) to identify the source of the problem. As Dr.María Luisa Caballero indicated, I am also not sure 4hr is necessary or not for your analysis...
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