Protein gel electrophoresis and western blotting

what are the 2 groups of methods for assessing protein purity

column chromatography methods, electrophoresis methods including SDS PAGE and 2DE

what is the basic method of assessing purity of proteins using SDS PAGE

1. proteins are denatured

2. treatment with SDS

3. voltage is applied, electrophore through pourus polyacrylamide gel

4. SDS treated proteins move to anode (+)

what does treatment with SDS do in SDS PAGE?

sodium dodecyl sulphate binds non-covalently to proteins giving an overall negative charge

what is the name of the gel that sieves proteins in SDS-PAGE?

a vertical acrylamide gel

what does the varying % in acrylamide gel mean?

different ones produce ‘lose’ or ‘tight’ mesh - one used depends on the denatured mwt’s of interest

which forces does denaturation distrupt?

the forces that stabalise secondary to quaternary structures

what is used for denaturation?

haotrophic agents e.g urea, B-mercaptoethanol, and SDS reducing agents

describe denaturation process

urea disrupts non-covalent interactions (only has to be used sometimes), B-mercaptoethanol used to reduce disulphide bonds, and then SDS unfolds protein into rigid rod and produces negative charge all along protein

what are the different staining methods that can be used to visualise proteins

coomassie blue, silver-strain, flourescent stains

what is western blotting used for?

used to detect and quantify a specific protein of interest in an exrtract/mixture, proteins are transferred out unstained gel after electrophoresis onto protein binding membrane using highly specific antibodies that are raised against protein of interest

describe basic method of western blotting

1. electrophoretically transfer fractionated proteins onto membrane

2. block membrane

3. incubate with primary antibodies specific to target protein

4. incubate membrane with HRP labelled secondary antibody specific to primary antibody

5. incubate blot with chemilluminescent HRP substrate and expose to film

what membranes can be used in western blotting?

PVDF or cellulose

what is the point of blocking the membrane in western blocking?

done to prevent unocupied sites on membrane preventing detection of antiodies binding non-specifically, which would cause background noise

what are the 2 dimensions to 2D gel electrophoresis?

isoelectic focusing (IEF) and SDS PAGE

explain isoelectric focusing in 2D gel electrophoresis

this separates proteins depending on their isoelectic points (pI), this is the pH at which particular protein has no net overall charge

describe method of isoelectic focusing

1. protein mixture is applied to gel strip containing immobilised pH gradient (IPG)

2. proteins migrate to pH where net charge is 0

3. at a pH equivalent to its pI, a protein has no overall charge and stops moving

what are 3 key applications of 2D gel electrophoresis

identifying disease biomarkers, diagnosis, and informing about disease mechanisms