SDS-Polyacrylamide Gel Electrophoresis

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From the 15/11/13 Immunology and Disease lab.

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Question Answer
What does SDS stand for? What is its purpose in this technique? Sodium dodecyl sulphate - this is a detergent which 'uncurls' proteins and coats them in a uniform negative charge, meaning that the only factor determining the distance travelled through the acrylamide gel is its size.
What is the point of the molecular weight markers? These are known proteins (of known molecular weight) so a standard curve of molecular weight plotted against distance travelled in the gel can be constructed in order to work out the molecular weight of unknown samples.
What is the difference between the stacking gel and the resolving gel? What is its significance? The stacking gel has a lower concentration of acrylamide, which means it forms larger pores when it cross-links with TEMED, so all the proteins in a given sample can travel through to the resolving gel. The resolving gel has a higher concentration of acrylamide to cross-link with TEMED, producing smaller pores which separate the proteins based on size throughout the gel. This allows the proteins to be separated.
What is the function of glycerol mixed in with the samples in the stacking gel? It is a viscous substance which weighs down samples when they are added to the sample wells. This prevents the samples from diffusing into the gel before the gel is run.
What is the function of the bromophenol blue dye added with the samples? This allows the progress of electrophoresis to be tracked. The dye molecules are smaller than any of the proteins and so they travel the fastest/furthest, allowing the movement of the proteins to be estimated/visualised.
Towards which electrode do the proteins travel? Why? They travel towards the positively charged anode because they are negatively charged.
Once the plate has been run, the bromophenol blue dye will have separated from the proteins. How are they visualised once again? Coomassie blue dye is applied to the plate. The plate is then de-stained, which means the remaining dye only stains the protein bands, allowing them to be accurately visualised.
What is the general process called when SDS-PAGE is combined with an ELISA in order to produce a 'print' of the electrophoresis gel? Western blotting.
When immunoglobulin samples are run on an electrophoresis plate, why are two protein bands produced? Because the proteins are separated into the light chains (which travel furthest) and the heavy chains.
What is a thick protein band indicative of? A lot of protein.
How many heavy, and how many light, chains does a pentameric immunoglobulin (IgM) have? 10 of each.
How many heavy, and how many light, chains does a dimeric immunoglobulin (e.g. IgA) have? 4 of each
How many heavy, and how many light, chains does a monomeric immunoglobulin (e.g. IgG) have? 2 of each
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