Hello
I don't understand the relation between reagents used in SDS-PAGE making.
I mean there are many protocols that say how to make SDS-PAGE, for example 15%, but they don't mention how they calculated the amount of reagents used to make these protocols.
for example how do they calculate the ratio of water and acrylamid-bisacrylamid amounts in SDS-PAGE 15% or other percentages?
i want to make my own SDS-PAGE but i don't know how calculate these amounts. i want to know the details about how to make it.
thanks in advance.
Here is one source of very detailed information on SDS-PAGE, including recipes:
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf
I would recommend that you buy pre-made acrylamide stock solutions, if you can afford to, in order to reduce the hazard of handling acrylamide, which is neurotoxic.
Dear Nii,
The following is the composition of SDS-PAGE:
For a 5 ml stacking gel (acrylamide percentage 4%):
H2O; 2.975 ml
0.5 M Tris-HCl, pH 6.8; 1.25 ml
10% (w/v) SDS; 0.05 ml
Acrylamide/Bis-acrylamide (30%/0.8% w/v); 0.67 ml
10% (w/v) ammonium persulfate (AP) ;0.05 ml
For this preparation you should use 0.67 mL of a solution mixture of Acrylamide/Bis-acrylamide that contains 30%w/v Acrylamide and 0.8% w/v /Bis-acrylamide.
For the preparing a 100 mL gel you multiply each component with 20.
The concentration of Acrylamide/Bis-acrylamide (30%/0.8% w/v) in this gel is:
20 x 0.67/100 = 13.4%, since the acrylamide solution used is 30% the final concentration of acrylamide will be about 4%.
For more examples, please use the following link:
http://www.assay-protocol.com/molecular-biology/electrophoresis/denaturing-page
Hoping this will be helpful,
Rafik
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