Thursday, 16 October 2014

First SDS PAGE of the year

Today we ran our first samples from the ammonium sulphate fractionation of milk proteins. The gels are looking fine, but I thought I would remind you of the key principles before we provide the class gels on the Y12 Edmodo site. The acronym, SDS stands for sodium dodecyl sulphate (LHS) and you may notice a resemblance to phospholipids: a long hydrophobic tail, capped by a negatively charged group. This molecule is added to the protein samples in order to stabilise the polypeptide chain in an extended (think of an extended, negatively charged rod, a little like DNA?). This renders most of the proteins identical apart from the length of the rod, which in turn is directly proportional to the protein's molecular weight.

The next part of the acronym, PAGE, stands for polyacrylamide gel electrophoresis. The polymerisation of acrylamide stimulated by TEMED and ammonium persulphate, provides us with a durable medium for the separation of a few hundred micrograms of proteins (of different molecular weights), which can be detected following a staining procedure involving Coomassie Blue, as seen RHS. The banding pattern provides a rapid and sensitive evaluation of the proteins in a sample and is generated as the proteins are separated on the basis of molecular weight differences in the denatured state. It has become a favourite method in all experimental Biology labs since it was pioneered by Laemmli, over 40 years ago (original paper). 

Today, we approached milk protein purification empirically and your samples will be as good as the care you took to prepare them. There may be carry over of ammonium sulphate, which can distort the bands, but we shall see soon enough. Some useful tips are:

1. Label and track all samples closely (remember your numbers 1-6 will look the same as those of your colleagues next session!).
2. Take care to add an equal volume of the gel loading buffer (it promotes protein unfolding). 
3. Load less than 40ul of your sample and steady the Gilson with one hand. Take your time!
4. Make sure you have recorded the details of each sample carefully and note the lanes on the gel, where your sample was applied.

Key Words SDS PAGE, protein separation, analysis electrophoresis

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