Uses Of Spectrophotometry

Question 1

What are the 6 uses of spectrophotometer?

Answer 1

1. determination of protein concentration - direct-A280nm
2. Determination of protein concentration - indirect e.g Bradford, biuret or Lowry
3. Enzyme activity measurements
4. DNA quantitation
5. Cell number (A450-600nm) of bacteria
6. Chemical detection of carbohydrates

Question 2

4.1 Protein quantitation can be achieved by two main techniques

Answer 2

Ultraviolet absorption methods (direct) and colorimetric assays (indirect)

Question 3

4.1 The main factors governing the choice of protein assay are

Answer 3

Amount of protein available to assay
Presence of interfering compounds
Protein concentration
Assay specificity
Ease and reliability of each assay

Question 4

True or false? On occasion protein quantitation facilitates detection of contaminating protein in a mixture

Answer 4

True

Question 5

4.1.1 uv absorption methods for the determination of protein concentrations can be performed directly on the sample of interest without

Answer 5

The addition of any reagent or incubation steps

Question 6

The relationship between protein concentration and absorbanceis

Answer 6

Linear

Question 7

Proteins mainly absorb light at two particular wavelengths (wavelength maxima)

Answer 7

200-210nm & 280nm

Question 8

Absorbance at 280nm is primarily due to

Answer 8

Aromatic ring structures of the amino acids tryptophan (W), tyrosine (Y), phenylalanine (f) and histidine (h)

Question 9

Proteins which contain little or none of these amino acids , particularly tryptophan, exhibit

Answer 9

Reduced absorbency at 280 nm

Question 10

1% w/v =

Answer 10

10mg /ml

Question 11

Bovine serum albumin (BSA) absorbency coefficient at 1% w/v and 1mg/ml at 280nm

Answer 11

6.3 in w/v and 0.63 in mg/ml

Question 12

Immunoglobulin g ( IgG) absorbency coefficient

Answer 12

13.8 and 1.38

Question 13

Chicken ovalbumin (oval) absorbency coefficient

Answer 13

7 and 0.7

Question 14

Some detergents as well as nucleicacids also absorb at 280mm which represents a limitation on

Answer 14

The use of absorbency measurement (280nm) for protein quantitation, however with co-measurement of absorbency at 260hm and 280mm the formula can be used to minimise the
Interference ave to nuclei acid presence since proteins due to absorb significantly at 260 nm

Question 15

What formula can be used to minimise the interference due to nuclei acid presence

Answer 15

Protein concentration (mg/ml) = 1.55A 280hm - 0.76A260nm

Question 16

Absorbency at 200 - 210 nm (peptide band absorbance) is generally confined to the detection of

Answer 16

Peptides (small proteins)

Question 17

Elution of peptides following separation by reversed -phase high performance chromatography (rp-hplc) is monitored by

Answer 17

Passing the elutant from the column through a flow-cell irradiated with light in the range 200 - 210 nm

Question 18

Protein quantitation via uv absorption does not result in protein destruction, hence the sample can be used for

Answer 18

Further analysis eg activity measurement

Question 19

Uv absorption quantitation of proteins measurements must be performed in

Answer 19

Quartz curettes since plastic absorbs at a wavelength less than 320 - 340 nm