Practical 1 Flashcards
Bradford assay
Estimate total protein in sample (compare to standard)
ELISA
Identify specific protein in complex mixture- quantitative, compared to standard plot
Non denaturing electrophoresis
Separation of proteins in gel by size and charge, protein remains functional
Denaturing electrophoresis
Separates proteins by size- dentures proteins to primary structure (multimeric proteins form more than one band)
Enzyme activity assay
Determines if enzymes are functional
Spectrophotometer
UV to visual light
Colorimeter
Visual light only
Beer Lambert Law
Absorbance= molar absorbance coefficient (M-1cm-1) x concentration (mol L-1 s-1) x cuvette length
Lactate to pyruvate
Lactate + NAD+ —> pyruvate + NADH + H+
What wavelength of light does NADH absorb?
340 nm
SDS
Sodium dodecyl sulphate (ionic detergent)
Cons of Bradford assay
Can’t identify specific proteins
Standards and sample incubated for same time
Samples need serial dilution- source of error
How to perform ELISA
- Well coated with an antibody (antiLDH) that recognises LDH subunit A
- Antibody captures LDH
- Excess washed away
- Antibody 2 that recognises subunit B is added
- Antibody 2 is covalently linked to enzyme catalysing conversion of substrate to coloured product
- Wash away excess antibody B
- Add substrate and read amount of coloured product in plate reader spectrophotometer
SDS PAGE summary
Proteins only have primary structure Disulphide and non covalent bonds broken Proteins coated in -ve charge Migrate from -ve to +ve charge Limitation on accurate estimate of Mr due to pore size in gel.
Pros of Bradford assay
Estimates total protein
Fast assay
Cheap
SDS PAGE Pros
Fast
Estimates Mr of polypeptides
Individual peptide Mr
Semiquantitative (comparison to standards)
SDS PAGE cons
Only estimates protein Mr (not quantitative for amount)
2 proteins may have same Mr- can’t distinguish
Western blot/ other method required for specific identification
Non Denaturing PAGE summary
Proteins retain 3D shape and functionality
Separation by charge and size
Gives true Mr as not denatured- forms 1 band even if multimeric
ELISA Pros
Cheap Reproducible Specific Sensitive Rapid
ELISA Cons
Appropriate antibody must be available
Samples need serial dilution to fall within standard curve range
