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SDS PAGE - how sds page work (Oct/13/2006 )

hello all
how does an sds page gel realy work?


Hi fozia!

A SDS-PAGE is a electrophoresis where you have two different gels.

The small collecting gel is usually made containing 4% acrylamide (so you have bigger pores there) and has a neutral pH (6,5).
The bigger seperating gel has a high percentage of acrylamide depending on the size of the proteins of interest. It also has a basic pH.

The proteins behave different in the pHs of the gels.
The buffer is basic. So the proteins that are linearised and negatively charged because of the SDS run to the positive anode. In the collecting gel is the first change of pH, which is the isoelectric point of Glycine - so Glycine that is in the loading buffer won't wander in the electric field because it is neutrally charged now. So you get a lower amount of charged particles what makes the electric field strength higher (because of rising electric resistance, U=R*I). Because of this the other still negatively charged proteins are accelerated and get concentrated (you get sharp bands).
In the seperating gel is basic pH again so the residual Glycine is getting charged again and runs out of the gel faster than the others, because now the proteins are seperated due to their size. The higher pecentage of acrylamide hinders bigger proteins to run fast, so the smaller ones reach the end faster. So if you like to seperate smaller proteins you chose a higher percentage of acrylamide to make them run slowly.

Well, I hope I didn't forget to mention some important things about the properties of SDS-PAGE... smile.gif



very nice introduction by Chakchel; I like to add that are some variations of the basic setting; beside described discontinuous SDS PAGE you may perform continuous f.i. if you like to separate very large proteins such as specific high range sized muscle proteins and then use a gradient of separation phase (e.g. 4-16%); you may also create a discontinuous gradient gel, e.g. 8-20% acrylamide in separation phase

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