In a tube we tried the following measurements to see if it the acrylamide would polymerize before we ran the gel:
1.15mL ddH20
1mL acrylamide
0.25mL NaH2PO4 buffer
330 uL APS
33 uL TEMED
Our protein of interest has a pI = 7.1. Every time the gel polymerizes, it is too watery when it needs to be more firm to be stable enough for our protein. Should I increase the acrylamide added to the solution? The above protocol is already a 10X of the original amounts specified for TEMED and APS. Maybe I should decrease the water?
Hi,
it depends on final acrylamide concentration. What is your stock AA conc? In theory, you can handle gels of about final 4% AA, however, it is a tricky business. Gels from 6% up (easy to handle can be considered everything above 8% ) are much easier to work with.
PS: Just to be sure, did you mix acrylamide with bis-acrylamide?
Thank you for answering Martin. I was not mixing my acrylamide with the bis-acrylamide! My P.I. just talked to me about this. I just started research so it may seem like a silly mistake. Thank you for your help :)
Hi,
You are right addition of bis-acrylamide is necessary in the polymerization. So far as the Native PAGE is concerned, the concentration of acrylamide will be determined only when you know the molecular weight of your protein of interest.
The concentration of acrylamide as suggested by Martin has a range and I believe you need to increase the concentration of acrylamide taking into mind the molecular weight of your protein of interest.
Hi Radiance,
Maybe this paper could help you, it explains BN- PAGE´s principles and I think it is a great way to be ready before even casting a gel.
http://www.ncbi.nlm.nih.gov/pubmed/17406264
Best regards
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