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Flashcards in Spectrophotometric Assays of Proteins Deck (17)
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1

What is protein assay?

Quantitation of protein concentration in:

- Cell extract
- Purified protein (MGH)
- Biological fluids (serum, plasma, etc.)

2

What are the units for protein concentration?

mg/mL (same as microg/microL but only for the same protein)

mM (mmol/L)

3

Why protein assay?

A required step before submitting protein samples for separation and analysis:

- Chromatography
- Electrophoresis
- Immunochemical separation or analyses
- Enzyme assays
- Protein-protein interactions

4

What are the two methods of spectrophotometric assays?

UV-based assays (280 nm max)
- Native colorless solution

Colorimetric assays
- Bradford assay (595nm)
- BCA assay (562nm)
- Colored protein sample

5

What is the basic structure of spectrophotometers?

Spectrometer first
1. Light source
2. Lens (Collimator)
3. Prism or Grating (monochromator)
4. Slit (Wavelength Selector)

Then photometer:
5. Sample solution (in cuvette)
6. Detector
7. Absorbance

6

What does spectrometric assay depend on?

Beer-Lambert law
A = ecL

Linear slope (good from 0.1 - 0.5 Abs)

Molecular crowding happens when it starts to curve from its linear slope

7

What is the relationship between absorbance and concentration?

Absorbance is directly proportional to concentration

8

What are the two ways to find the concentration of a protein solution?

Published extinction coefficient
C=A/eL
- Good for UV-based assays
- Purified proteins

Standard Calibration curve of BSA
- A~C
- Linear slipe (e) = A/C
- Good for colorimetric assays
- Purified proteins

9

What is a extinction coefficient?

Light absorptivity (e)

A measure of how strongly a chemical species or substance absorbs light at a particular wavelength (wavelengthmax)

10

What is an intrinsic property?

Dependent upon their chemical composition and structure

11

How to find molar extinction coefficient of a folded protein with the use of amino acid composition?

(5500 x nTrp) + (1490 x nTyr) + (125 x nS-S (Cysteine))

12

What are the advantages and disadvantages of UV-based Assay?

Advantages:
- Quick and simple, no assay reagents required
- Non-destructive (can be reused)

Disadvantages:
- Highly error prone with protein mixtures or complex samples (cell lysates)
- Expensive (quartz cuvette)
- Molar extinction coefficient not known for many proteins

13

If amino acid composition is not known, how can protein concentration be calculated with absorbance?

1.55 (A280) - 0.76 (A260)

14

What are the advantages and disadvantages of Coomassie Blue Dye-Binding Assay (Bradford method)?

Advantages:
- Very quick

Disadvantages:
- Not good for small proteins

Binds to positively charged amino acids (Arg, His, Lys)

Also interacts with aromatic AAs (Trp and Tyr) through hydrophobic interactions

15

What are the advantages and disadvantages of BCA Assay?

Advantages:
- Good for all kinds of proteins
- Good linearity over a wide range and high sensitivity

Disadvantages:
- Time consuming

Cysteine, Cystine, Trp, Tyr

16

What is the sensitivity?

Lower detection limit, Extinction coefficient equivalent

Sensitivity = A/cL

More light = more absorption

17

What is the relationship between absorption and path length?

When path length increases, absorbance increases