Assessing protein purity PDF

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Assessing protein purity Why do we need SDS PAGE? - As a tool in purification: To show the purity of proteins for: Enzymes assays, structural biology, biophysics, sequence determination - For identification purposes: To separate proteins according to size and observe differences between samples *SEE LAB PREP NOTES ON SDS PAGE AND COOMASSIE STAINING Silver staining • When Coomassie staining is not sensitive enough you can use Silver stain. • Again, gels are fixed but then exposed to silver nitrate which likely reacts with basic and thiol groups. • Reduction of silver leaves black/brown deposits. • Sensitivity = time and expense. Calibration of molecular weight marker To calculate the relative mobility (Rf)~ • Measure the distance each of the standards and your samples have travelled from the top of the separating gel. • Then dividing this distance by the distance travelled by the dye front.

Rf

=

migration distance of marker Migration distance of dye front

-Can plot this against molecular weight - log molecular weight for straight line and use line equation to determine molecular weight of unknown sample SDS-PAGE summary • Proteins are denatured by SDS and gain negative charge. • Proteins are separated by PAGE on the basis of size alone. • Gels are stained to reveal the presence of the proteins. • Protein size can be estimated in relation to molecular weight markers (proteins of known mass). SDS Gels In Practice: loading Buffer • Buffer e.g. Tris pH 6.8. – Keeps proteins stable. • SDS – Coats proteins – charge. • Glycerol. – Increases density. • Bromophenol blue. – Allows you to track progress. • B-mercaptoethanol – Reduces disluphide bonds.

SDS Gels In Practice: Acrylamide percentages • Increasing acrylamide percentage resolves smaller proteins better. – Opposite is also true. • Can also run gradient gels for even better separation SDS Gels In Practice: SDS-PAGE gels • Stacking gel – compresses samples. – Lower acrylamide content. – Lower pH (6.8). – Different ionic content. • Resolving gel – separates proteins by size. • Buffer: – Influences migration patterns. – Common buffers Tris, Bis-Tris and imidazole. • Counterion: – Balances the intrinsic charge of the buffer ion. – Normally glycine or tricine. • Acrylamide (C3H5NO): – Forms polymer chains in water. • Bisacrylamide (N,N'-Methylenebisacrylamide; C7H10N2O2): – Crosslinker of acrylamide. • Sodium Dodecyl Sulfate (SDS; C12H25NaO4S): – Coats protein with negative charge. • Ammonium persulfate (APS; N2H8S2O8): – Source of free radicals. – Used as an initiator for gel formation. • TEMED (N, N, N', N'-tetramethylethylenediamine; C6H16N2). – Stabilizes free radicals and improves polymerization. SDS-PAGE gels – pouring a gel

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Can also buy precast gels Precast gels – time = money

Advanced PAGE techniques - Isoelectric focussing o Relies on the pl of the protein o Electrophoresis technique that separates proteins based on their pl o Uses medium with a pH gradient o Voltage applied o Proteins migrate to their pl Studying the protein content • Firstly, select your cells / tissues / organisms. • Usually done as a comparison e.g. drug-treated vs. untreated cells. • Looking for differences in protein content between the two conditions. • Proteins may be increased (upregulated) or decreased (downregulated) as a response to conditions 2D gel electrophoresis - theory

Technique for purifying individual proteins from complex samples based on their pl AND molecular weights

2D gel electrophoresis - practice

Difference gel electrophoresis (DIGE) • Modification of 2D SDS-PAGE. • Each sample is tagged with a different fluorophore, combined, and run on a single gel. • Analysed by fluorescence.

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Identifying your proteins Achieved via mass spec afer cutng out of spot. Protein complexes – native gels • Native or non-denaturing gels. • Mobility depends on charge and hydrodynamic radius. • Changes in size therefore affect mobility. – changes in charge due to chemical degradation (e.g deamidation). – unfolded, "molten globule", or other modified conformations. – oligomers and aggregates (both covalent and non-covalent). – binding events (protein-protein or protein-ligand). • Electrophoretic mobility is difficult to predict. • Proteins can be re-natured in some cases and used to show activity (Zymogram)...