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Biochemistry 3380 > Lecture 5 > Flashcards

Lecture 5 Flashcards

1
Q

What reference protein did we use to create a standard curve?

A

bovine serum albumin (BSA)

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2
Q

What does the biuret assay measure?

A

nitrogens in peptide bonds

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3
Q

How does the biuret assay perform its measurements?

A

Cu2+ is converted to Cu1+ and the new complex absorbs more light energy at 550 nm

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4
Q

What are the advantages of the biuret assay? (2)

A

amino acid composition of proteins is not important; little interference by free amino acids

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5
Q

What is the disadvantage of the biuret assay?

A

Tris and NH3 interfere and inflate absorbance values

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6
Q

What does the A260/A280 assay measure?

A

measures absorbance of protein-containing solutions at 280 nm

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7
Q

Proteins lacking what proteins cannot be measured at 280 nm?

A

tryptophan, tyrosine, phenylalanine

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8
Q

What are the advantages of the A260/A280 assay? (2)

A

rapid; does not destroy sample

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9
Q

What are the disadvantages of the A260/A280 assay?

A

must be free of other compounds that absorb at 280 nm (like nucleic acids)

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10
Q

What is the equation used for the A260/A280 assay?

A

protein (mg/mL) = 1.55A280 - 0.76A260

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11
Q

What does the Lowry assay measure?

A

protein determination

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12
Q

What are the advantages of the Lowry assay? (4)

A

cheap; reproducible; sensitive; easy to perform

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13
Q

What are the disadvantages of the Lowry assay? (3)

A

sensitive to contaminants; standard curves are linear only at low protein concentrations; timing and mixing of reagents must be precise

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14
Q

What is the Lowry reaction?

A

consists of Biuret reaction followed by reduction under alkaline conditions of the Folin-Ciocalteu reagent

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15
Q

What is the absorption maximum of the Lowry product?

A

750 nm

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16
Q

The binding of the Bradford dye causes what?

A

shift in absorption maximum of dye from 465 nm to 595 nm

17
Q

How does the Bradford dye interact with the protein?

A

forms strong, noncovalent complexes with proteins via electrostatic interactions w/ amino and carboxyl groups via van der Waals forces

18
Q

What are the advantages of the Bradford assay?

A

one-step; sensitive; accurate

19
Q

What are the disadvantages of the Bradford assay?

A

dye stains cuvettes; high concentrations of detergents can interfere with this assay

20
Q

Scratches on the cuvette surface will cause

A

reflection

21
Q

Air bubbles in the cuvette will cause

A

scattering

22
Q

Oil/fingerprints on the cuvette surface will cause

A

absorption

23
Q

Write out the Beer-Lambert equation and identify the terms.

A

slide 19

24
Q

What is the relationship between concentration and absorbance in the context of Beer-Lambert’s law?

A

as concentration increases, absorbance increases (not valid at high concentrations)

25
Q

What is the color shift when the Bradford dye is added to the washes/eluates?

A

reddish brown to dark blue

26
Q

Given the conditions of an enzyme assay, be able to describe what components of that assay should be placed in the reference cuvette to properly blank the machine

A

Everything except the enzyme should be put in the blank cuvette, then BTV with sufficient dd H2O.

27
Q

Define wavelength.

A

the length of a full cycle of a wave (the distance between two peaks).

28
Q

Define wavelength scan.

A

looks at the absorption at multiple wavelengths

29
Q

Define maximum wavelength.

A

the wavelength at which you will get the maximum absorbance for a substance

30
Q

Define absorbance.

A

the amount of light that is blocked or captured by the substance which can be measured as a percentage

31
Q

Define transmittance.

A

the amount of light that is not absorbed by the sample

32
Q

What are the principle difference between a spectrophotometer and a spectrafluorometer?

A

photometer measures absorbance, whereas fluorometer measures fluorescence

33
Q

Define specific activity.

A

ratio of active protein to total protein

34
Q

What are the two common procedures for quantifying protein concentration that we used in this week’s laboratory?

A

Bradford assay; A280/A260 assay

Biochemistry 3380 (9 decks)

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