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Mims > Practical 1 > Flashcards

Practical 1 Flashcards

1
Q

Bradford assay

A

Estimate total protein in sample (compare to standard)

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2
Q

ELISA

A

Identify specific protein in complex mixture- quantitative, compared to standard plot

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3
Q

Non denaturing electrophoresis

A

Separation of proteins in gel by size and charge, protein remains functional

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4
Q

Denaturing electrophoresis

A

Separates proteins by size- dentures proteins to primary structure (multimeric proteins form more than one band)

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5
Q

Enzyme activity assay

A

Determines if enzymes are functional

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6
Q

Spectrophotometer

A

UV to visual light

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7
Q

Colorimeter

A

Visual light only

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8
Q

Beer Lambert Law

A

Absorbance= molar absorbance coefficient (M-1cm-1) x concentration (mol L-1 s-1) x cuvette length

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9
Q

Lactate to pyruvate

A

Lactate + NAD+ —> pyruvate + NADH + H+

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10
Q

What wavelength of light does NADH absorb?

A

340 nm

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11
Q

SDS

A

Sodium dodecyl sulphate (ionic detergent)

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12
Q

Cons of Bradford assay

A

Can’t identify specific proteins
Standards and sample incubated for same time
Samples need serial dilution- source of error

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13
Q

How to perform ELISA

A
  1. Well coated with an antibody (antiLDH) that recognises LDH subunit A
  2. Antibody captures LDH
  3. Excess washed away
  4. Antibody 2 that recognises subunit B is added
  5. Antibody 2 is covalently linked to enzyme catalysing conversion of substrate to coloured product
  6. Wash away excess antibody B
  7. Add substrate and read amount of coloured product in plate reader spectrophotometer
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14
Q

SDS PAGE summary

A 
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.
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15
Q

Pros of Bradford assay

A

Estimates total protein
Fast assay
Cheap

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16
Q

SDS PAGE Pros

A

Fast
Estimates Mr of polypeptides
Individual peptide Mr
Semiquantitative (comparison to standards)

17
Q

SDS PAGE cons

A

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

18
Q

Non Denaturing PAGE summary

A

Proteins retain 3D shape and functionality
Separation by charge and size
Gives true Mr as not denatured- forms 1 band even if multimeric

19
Q

ELISA Pros

A 
Cheap
Reproducible
Specific
Sensitive 
Rapid
20
Q

ELISA Cons

A

Appropriate antibody must be available

Samples need serial dilution to fall within standard curve range

Mims (6 decks)

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