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SDS-PAGE: Effect of Acrylamide concentration


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#1 Luria Bertani

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Posted 11 May 2011 - 07:33 PM

Hi there.

I've been doing SDS-PAGE from a set protocol, but I've noticed different groups around using different % of acrylamide.
Our lab has both large MW protiens (100 kDa and above) and also small MW proteins (25 kDa).

I was wondering what the relationship between the % Acrylamide and the resolution of proteins was? For example, does a higher % of Acrylamide make small MW proteins resolve bands better at the expense of worse resolution of high MW protein bands?

Thank you in advance

LB

#2 protolder

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Posted 11 May 2011 - 09:53 PM

Hi there.

I've been doing SDS-PAGE from a set protocol, but I've noticed different groups around using different % of acrylamide.
Our lab has both large MW protiens (100 kDa and above) and also small MW proteins (25 kDa).

I was wondering what the relationship between the % Acrylamide and the resolution of proteins was? For example, does a higher % of Acrylamide make small MW proteins resolve bands better at the expense of worse resolution of high MW protein bands?

Thank you in advance

LB

Hola, as you know acrylamide is as an obstacle in the proteins race.As more concentrated acrylamide is more difficult for proteins to cross throught it, but always little ones will run faster than biggers.So in a 15% gel all have "problems" to run but the resolution is better between the small ones, and all the big remain together in the top of the gel. In the other hand diluted gels made that all the smalls run together without resolution and the big ones separate better. Look information about protein markers because in any of them (bio_Rad , amersham or any other) has examples of how the resolution is in different acrylamide %.In your case I think that a 10% could resolve both areas of your proteins. One more thing some people make in gradient gels to improve resolution. Buena suerte

#3 Luria Bertani

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Posted 12 May 2011 - 12:54 AM

Thank you, so helpful!

LB.

May your protein yields be high...




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