Module 6

Lyophilize

Freeze-dry and remove water from proteins

Weigh on balance

Proteins misfold and lose activity

UV/Vis Spectrophotometry Drawbacks

Not for 2+ proteins

Not for samples with DNA

Differential UV Absorbance

Benzene ring in aromatic amino acids absorb UV

More rings = more absorption

Colourimetric Assay

Biochemical test with colour-changing sample

Measure molecule concentration

Measure enzyme activity

Bradford Assay

Colourimetric

Measure coomassie blue colour change when bound to protein

Red unbound, blue bound (595 nm)

Overestimate proteins with more positive and aromatic amino acids

Bicinchoninic Acid Assay (BCA)

Colourimetric

Measure Cu+ from protein reduction and BCA complex

Purple complex (560 nm)

Overestimate proteins with cysteine, tyrosine, tryptophan

Spectrophotometry: DNA Influence

Small impact

No interaction with coomassie and BCA

Spectrophotometry: Buffer Influence

Small impact

NO interaction with coomassie and BCA

Spectrophotometry: EDTA

Remove metal ions from proteins

Bind Cu2+ (not for BCA)

Calibration Curves

Interpret colourimetric assay results

BSA protein standard of known concentration

Line of best fit for absorbance vs protein concentration

Calibration: BCA Assay

Sensitive

Wide range

For high and low concentrations

Calibration: Bradford Assay

Rapid

Less sensitive

Wide range

For high concentration only

Calibration: Reduce Error

Same time intervals

Measure replicates

Colourimetric Assay Microplates

More efficient

Use multichannel pipette

Read with microplate reader

SDS-PAGE

Separate proteins by size

Analyze protein mixture

SDS-PAGE Gel

Acrylamide and bisacrylamide

Bilayer

Top layer: low %

Bottom layer: high %

High resolution

SDS-PAGE Gel %

Low: Large pores, faster protein travel

High: Small pores, slower protein travel

SDS-PAGE: SDS

Negative solution

Boil with protein

Denature and coat protein with negative charge (pull to anode)

SDS-PAGE: Buffer

SDS denature and coat proteins

Glycerol increase density

Dye visualization

Reducing agent break disulfide bonds

Western Blot

Detect and quantify protein in complex sample

Western Blot: Antibodies

Monoclonal IgG

Bind antigen epitope

Western Blot Process

1. Transfer SDS proteins to membrane with electric field
2. BSA wash prevent non-specific antibody binding
3. Add primary antibody
4. Add enzyme-linked secondary antibody
5. Add detection substrate
6. Measure light emission proportional to protein amount

Western Blot Specificity

Antibody recognize specific protein

Identity and size

Western Blot Sensitivity

Very sensitive

More than absorbance and colourimetric measurements

Coomassie Assay vs Western Blot

Coomassie: Determine size and purity, no identity

Western: Determine identity and size, no purity

ELISA

Detect protein presence and amount in mixture

Direct ELISA

Detect antigens

Analyze immune response

Indirect ELISA

Detect and quantify antibodies

Sandwich ELISA

Detect and quantify antigen protein

Competitive ELISA

Detect small antigens

1 antibody

Sandwich ELISA Process

1. Add capture antibody
2. Add sample
3. Add detection antibody
4. Add enzyme-linked antibody
5. Add substrate
6. Read microplate (colour change/light emission proportional to protein amount)

ELISA vs Western Blot

ELISA: Use enzyme-linked antibodies, no protein separation and membrane

Western: Use enzyme-linked antibodies, protein separation and membrane