Methods of Protein Detection

Question 1

Which range of the light spectrum is used in protein photospectrometry?

Answer 1

UV - visible

Question 2

Which absorbance is generally used?

Answer 2

280nm

Question 3

When would a colorimetric method be used?

Answer 3

In the detection of prosthetic groups

Question 4

What is the principle of the Biuret method?

Answer 4

Biuret method employs protein’s reaction with an alkaline solution of copper-sulphate to produce a violet complex, essentially detecting peptide bonds. The shade of the colour indicates the proportion of protein. The absorption maximum is 545nm.

Question 5

List the advantages of the Biuret method.

Answer 5

Automated prospects, simple and reliable, and can be scaled down, to consume less protein

Question 6

List the limitations of the Biuret method.

Answer 6

Lack of sensitivity, consumes large amounts of sample, and a standard curve is requisite for it use

Question 7

Name the predominate, and subordinate, amino acids that are detected by the Lowry method.

Answer 7

Tyrosine, and tryptophan

Question 8

What colour complex is produced by the Lowry method?

Answer 8

Blue

Question 9

Outline the principle of the Lowry method,

Answer 9

Poorly understood, but it appears to be similar to the Biuret method. The commercially-produced Folin-Ciocalteau reagent contains phosphomolybdic acid and phosphotungstic acid, to produce a further blueish complex

Question 10

What are the advantages and disadvantages of the Lowry method?

Answer 10

Very sensitive, with broad absorption peaks, but requires calibration, and results are proportionate to amount of tyrosine and tryptophan present.

Question 11

By which other name is the Biochinonic Acid (BCA) assay known?

Answer 11

Pierce Assay

Question 12

Which biochemical apparatus is used in the BCA assay?

Answer 12

Microplate

Question 13

From which method is the BCA assay adapted?

Answer 13

Lowry

Question 14

What dye is used in the BSA assay?

Answer 14

Coomassie Brilliant Blue

Question 15

At which absorbency is the BSA assay measured?

Answer 15

595nm

Question 16

What is an advantage and disadvantage to the BSA method?

Answer 16

Very sensitive, but reagents used are unstable, thus having the potential to give erroneous results.

Question 17

What can intrinsic absorbance be used for?

Answer 17

To detect the presence of the protein, determine the concentration of the protein, and take a spectrum, to determine if different forms of the protein are present

Question 18

Where is rhodopsin found, what is it for, where in the spectrum does it absorb, and what is its protein known as?

Answer 18

Found in the rods of the eye, and it is responsible for night vision. It strongly absorbs in the green-blue region of the spectrum, with a maximum absorption of 495 nm. It appears as a reddish-purple hue, and its protein is known as visual purple

Question 18

What is Cytochrome C552, and what colour is it?

Answer 18

It is the electron carrier in the electron transport chain, and is red, due to its possession of haem c.