We are optimizing SDS-PAGE, i would like to know the best ratio and percentage for the Staking gel and separation gel of Acrylamide.
Its like 37.1:1 and 29.1 and 19.1 and also 30% and 40%,
So i am confused about it.
Your recommendation would help alot.
Hi
You could use 4% and 15% stacking and resolving gels, respectively. Moreover, you could find more details in Sambrook and Russels (2001) reference book.
Acrylamide: Bis-Acrylamide Ratio of 37.5:1 is good for protein gels and 19:1 is good for DNA Urea PAGE gels. I haven't personally tried the 29:1 ratio.
You need a discontinuous gel for good separation of the protein ladder. 5% stack and 12 or 15% separating gel is optimal for your protein of interest.
Alternatively, you could do even better and make a 5 - 15% gradient gel. You just need a serological pipette, solutions for polymerization of the stacking and separating gel and you mix the two solutions with a single air bubble.
https://www.youtube.com/watch?v=zu5a-kpMK8k
Typically a 12% Acrylamide Gel will work fine, however a 10% Acrylamide gel would be more ideal for the range you are referring to.
Use 30% acrylamide and make 10 to 12% resolving gel. Use 4% stacking gel with 30% acrylamide. You will get sharp band.