I am attempting to seperate out protein fragments from various snake venoms using gel electrophosesis. I know that electrophosresis runs from negative to positive and that proteins are positively charged and so therefore need to be denatured and treated to reverse that polarity. I understand that sodium dodecly sulfate will do this, but I can't find information on how to use it correctly (a procedure). If anyone could help me out with that, I would greatly appreciate it!
- Christian
Protein preparation for agrose gel electrophoresis
Moderators: AmyCowen, kgudger, MadelineB, Moderators
Re: Protein preparation for agrose gel electrophoresis
I have an electrophoresis protocol that I use in my molecular biology class, but it is a rather long protocol.
I extract proteins from cells using the following extraction buffer: 6 M urea, 3% high quality SDS, 6% β-mercaptoethanol, 0.003% bromophenol blue). I pellet the cells using a centrifuge, then lyse the cells by resuspending the pellet in 50 ul of extraction buffer and heating the mixture at 65oC for 10 minutes. If you are extracting proteins from cells, you might also want to have a TB syringe with a needle to aspirate the lysate several times after heating to break up the DNA which can make the lysate really goopy. Since it appears you are not using cells as your starting material but rather snake juice (lol), maybe mix the snake juice 1:2 with the extraction buffer and then heat it. If you can't run the proteins right away, freeze them ASAP after heating. Good luck!
I extract proteins from cells using the following extraction buffer: 6 M urea, 3% high quality SDS, 6% β-mercaptoethanol, 0.003% bromophenol blue). I pellet the cells using a centrifuge, then lyse the cells by resuspending the pellet in 50 ul of extraction buffer and heating the mixture at 65oC for 10 minutes. If you are extracting proteins from cells, you might also want to have a TB syringe with a needle to aspirate the lysate several times after heating to break up the DNA which can make the lysate really goopy. Since it appears you are not using cells as your starting material but rather snake juice (lol), maybe mix the snake juice 1:2 with the extraction buffer and then heat it. If you can't run the proteins right away, freeze them ASAP after heating. Good luck!