Methods of Protein Detection Flashcards
Which range of the light spectrum is used in protein photospectrometry?
UV - visible
Which absorbance is generally used?
280nm
When would a colorimetric method be used?
In the detection of prosthetic groups
What is the principle of the Biuret method?
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.
List the advantages of the Biuret method.
Automated prospects, simple and reliable, and can be scaled down, to consume less protein
List the limitations of the Biuret method.
Lack of sensitivity, consumes large amounts of sample, and a standard curve is requisite for it use
Name the predominate, and subordinate, amino acids that are detected by the Lowry method.
Tyrosine, and tryptophan
What colour complex is produced by the Lowry method?
Blue
Outline the principle of the Lowry method,
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
What are the advantages and disadvantages of the Lowry method?
Very sensitive, with broad absorption peaks, but requires calibration, and results are proportionate to amount of tyrosine and tryptophan present.
By which other name is the Biochinonic Acid (BCA) assay known?
Pierce Assay
Which biochemical apparatus is used in the BCA assay?
Microplate
From which method is the BCA assay adapted?
Lowry
What dye is used in the BSA assay?
Coomassie Brilliant Blue
At which absorbency is the BSA assay measured?
595nm
What is an advantage and disadvantage to the BSA method?
Very sensitive, but reagents used are unstable, thus having the potential to give erroneous results.
What can intrinsic absorbance be used for?
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
Where is rhodopsin found, what is it for, where in the spectrum does it absorb, and what is its protein known as?
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
What is Cytochrome C552, and what colour is it?
It is the electron carrier in the electron transport chain, and is red, due to its possession of haem c.
