![]() ![]() The nitrocellulose is then soaked in gelatin to "block" its ability to non-specifically bind proteins.After this the gel is placed over a sheet of nitrocellulose and the protein in the gel is electrophoretically transfered to the nitrocellulose.A protein sample is subjected to polyacrylamide gel electrophoresis.This method usually involves the following steps: quantitative (you need an accurate way to measure the quantity of your protein at each step in the purification)Īntibodies can be used in a method called Western blotting, which is useful for determining levels of protein expression and for assaying proteins during purification.sensitive (you don't want to consume all your sample in order to assay it).rapid (you don't want to wait a week for the results).Specific (you don't want a false positive).molecular mass, spectroscopic properties, etc.) The specific assay can be based upon some unique characteristic of the protein of interest ![]() The first step in any purification is the development of a specific assay for the protein of interest. from which you may have to extract milligram (or microgram!) quantities of desired protein at high purity, and hopefully with high yield. coli, or trying to isolate a protein from some mammalian tissue, you are typically starting with gram quantities of a complex mixture of protein, nucleic acids, polysaccharide, etc. Whether you are starting off with a recombinant protein which is produced in E. Running the Experiment, Resolving PeaksĪssays, Specific Activity, Initial FractionationĪ successful protein purification procedure can be nothing short of amazing.Gel Filtration, Affinity and Hydrophobic resins Preparation of Resin, Plumbing.Elution of proteins from ion exchange resins.Column Chromatography - Ion exchange Dialysis and Concentration.Assays, Specific Activity, Initial Fractionation. ![]()
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