I accidently added double volume of bisacrylamide i.e: 1.6 instead of 0.8 during the preparation of solution. Will it effect the sds page or protein bands? Please suggest me. Thank You..........
yes, the gel will be more cross-linked. You could add the water and acrylamide to take the concentration of bisacrylamide to the usual level.
The pore size of the gel, and the separation/resolution of protein will be different. You could refer to BioRad Electrophoresis guide available online in Chapter 4: Reagent selection and preparation about the % of acrylamide.
If u are making a Acrylamide stock, then double all other constituents except Bis-acrylamide. Ur Problem is solved.
The pore size of the gel will change and so will the resolution of bands. just double all other components in the solution except for bis-acrylamide to bring the concentration back to the desired level.
Thanks to all for your suggestions. I have now doubled the volume of acrylamide to balance the solution. Thank you once again.
I agree to the above comments and would like to say,It could decease your gel pour size and your sample will need more time for running. additionally, your resolution would be better but it depends on low voltage which will waste your time to wait for more hours. Simply, you can balance the concentration by increasing the volume.
Dear Sobia,
Altering the ratio of acrylamide to bis-acrylamide can have complex effects. I would advise running the gel anyway if you have sample to spare, side-by-side with a gel cast with your usual recipe; you may discover something interesting in the way your protein of interest migrates.
I am also reposting this informative link from another thread:
https://nationaldiagnostics.com/electrophoresis/article/polyacrylamide-matrix
These are relevant paragraphs in that article.
"For discussions of the composition of polyacrylamide gels, a standard nomenclature has been widely adopted. In this nomenclature, T represents the total percentage concentration (w/v) of monomer (acrylamide plus crosslinker) in the gel. The term C refers to the percentage of the total monomer represented by the crosslinker. For example, an 8%, 19:1 (acrylamide/bisacrylamide) gel would have a T value of 8% and a C value of 5%."
"Control of the pore size of a polyacrylamide gel is accomplished by changing the T and C values. With increasing T, the pore size decreases in a nearly linear relationship. Higher percentage gels (higher T), with smaller pores, are used to separate smaller molecules. The relationship of C to pore size is more complex. Generally, the minimum pore size occurs when C is about 5% (a 19:1 gel). Decreasing C results in a more open pore structure because there are fewer crosslinker molecules. Increasing C beyond 5% also increases the pore size. This appears to be because of nonhomogeneous bundling of strands in the gel."
Thanks to all specially to Mark K Chee whose suggestion helped me a lot.
The effect ot %T and %C on pore size was investigated in
@article {Ste-98
author = {Stellwagen, N.C.},
title = {Apparent pore size of polyacrylamide gels: Comparison of gels cast and run in Tris-acetate-EDTA and Tris-borate-EDTA buffers},
journal = {ELECTROPHORESIS},
volume = {19},
number = {10},
doi = {10.1002/elps.1150191004},
pages = {1542-1547},
year = {1998},
}
Can separation resolution can be changed if the amount of polyacrylamide in the gel is changed?
Of course.If you run the sample sample in gels of 10 %, 12% and of 15 % you may observe difference in the resolution as well. But if you want to enhance your band resolution you may use Tricine-PAGE specially designed for low molecular weight proteins.
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