Thursday Jan 29 2009
This lab should have taken place on Wednesday Jan 28, but classes were cancelled due to weather.
Polyacrylamide Gel Electrophoresis (PAGE) is a technique used for separating polynucleotides or polypeptides that are very similar in size, providing greater resolution than with an agarose gel.
We used PAGE to measure the size of a protein, GFP. If we follow the series of "make believe" in which we are dealing with a new protein, this would be a step to find more information about it. Protein size is quantified in Daltons (Da), a measure of molecular mass. One Dalton is defined as the mass of a hydrogen atom, which is 1.66 x 10-24grams (g).
PAGE is used for separating proteins ranging in size from 5 to 2,000 kDa due to the uniform pore size provided by the polyacrylamide gel. Agarose gels can also be used to separate proteins, but they do not have a uniform pore size, so they are optimal only for electrophoresis of proteins that are larger than 200 kDa. We ran two types of PAGEs: Native and denaturing (a.k.a. SDS)
Proteins can have varying charges and complex shapes, therefore they may not migrate into the gel at similar rates, or at all. In native gel electrophoresis the proteins being separated differ in molecular mass and intrinsic charge and experience different electrophoretic forces dependent on the ratio of the two. Because of different charges and tertiary structure proteins of the same mass may migrate at different rates.
In SDS gel electrophoresis proteins are denatured (linearized) in the presence of a detergent such as Sodium Dodecyl Sulfate (SDS) that coats the proteins with a negative charge. The resulting denatured proteins have an overall negative charge, and all the proteins have a similar charge to mass ratio. Since denatured proteins act like long rods instead of having a complex tertiary shape, the rate at which they migrate in the gel is relative only to its size (molecular weight) and not its charge or shape.
We will be able to compare teh results in both gels, and if GFP has any activity in either one of them (through pictures taken under UV light).
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