Methods for Total Protein

Question 1

Enumerate the Methods for Total Protein

Answer 1

1. Kjeldahl Method
2. Biuret Method
3. Folin-Ciocalteu (Lowry Method)
4. UV Absorption Method
5. Refractometry
6. Turbidimetry and Nephelometry
7. Coomasie Brilliant Blue Dye
8. Ninhydrin
9. Serum Protein Electrophoresis (SPE)

Question 2

• Classical method for protein quantitation
• Reference method but not routinely used because it is very time consuming and only uses an assumption
• Not accurate

Answer 2

Kjeldahl Method

Question 3

T/F. In Kjeldahl Method, Measures the amount of nitrogen in specimen (Protein = CHON).

Answer 3

True

Question 4

• Assumes the nitrogen content of protein is 16% (although, it varies between 15.1%-16.8%)

Answer 4

Kjeldahl Method

Question 5

In Kjeldahl Method, 1g of nitrogen is equal to

Answer 5

6.54% of protein

Question 6

3 steps: In Kjeldahl Method

Answer 6

Digestion, Distillation, Titration

Question 7

• A colorimetric, non-enzymatic method
• Most widely used method for total protein determination

Answer 7

Biuret Method

Question 8

Biuret Method End product:

Answer 8

Violet-colored

Question 9

Biuret Method Absorbance of color is read at

Answer 9

540nm

Question 10

Measures peptide bonds present in protein forms a bond with cupric ions forming a violet-colored chelate

Answer 10

Biuret Method Principle

Question 11

T/F. In Biuret Method, More peptide bonds = Dark color = More protein

Answer 11

True

Question 12

Biuret Method Interference:

Answer 12

Lipemic sample

Question 13

Enumerate the Biuret reagent

Answer 13

o Alkaline CuSo4
o NaK tartrate (Rochelle salt) – to prevent precipitation of copper
o NaOH
o KI – stabilizer

Question 14

Has the highest analytical sensitivity and can measure even the smallest amount of protein

Answer 14

Folin-Ciocalteu (Lowry Method)

Question 15

Oxidation of phenolic compounds such as tyrosine, tryptophan and histidine to give a deep blue color (measures spectrophotometrically)

Answer 15

Folin-Ciocalteu (Lowry Method) Principle

Question 16

Folin-Ciocalteu (Lowry Method) Main reagent:

Answer 16

Phosphotungstic-Molybdic acid or phenol reagent

Question 17

T/F. Phosphotungstic-Molybdic acid or phenol reagent, can oxidized phenolic compounds

Answer 17

True

Question 18

Color enhancer use in Folin-Ciocalteu (Lowry Method)

Answer 18

Biuret reagent

Question 19

The absorbance of proteins at 210nm is due to the absorbance of the peptide bonds at specific wavelength

Answer 19

UV Absorption Method Principle

Question 20

T/F. In UV Absorption Method, Most proteins has 210nm absorption

Answer 20

True

Question 21

Absorption at 280nm:

Answer 21

Aromatic AA (Tryptophan, tyrosine and phenylalanine)

Question 22

• Based on measurement of refractive index of serum total proteins
• Based on how protein bend light which is directly proportional (Increase changes = More protein)

Answer 22

Refractometry

Question 23

Measurement depends on formation of a uniform fine precipitate which scatter incident light in suspension (nephelometry) or block light (turbidimetry)

Answer 23

Turbidimetry and Nephelometry

Question 24

Used for detection of proteins as little as 1ug

Answer 24

Coomasie Brilliant Blue Dye

Question 25

Develops violet color by reacting with primary amines widely used for detection of peptides and amino acids after chromatography

Answer 25

Ninhydrin

Question 26

it is a type of separation technique

Answer 26

Serum Protein Electrophoresis (SPE)

Question 27

Migration of charged particles in an electric field

Answer 27

Serum Protein Electrophoresis (SPE) Principle

Question 28

Negative terminus →

Answer 28

cathode

Question 29

Positive terminus →

Answer 29

anode

Question 30

T/F. In (SPE), Isoelectric property of proteins (they go to anode)

Answer 30

True

Question 31

either positive or negative depending on pH condition

Answer 31

Amphoteric

Question 32

(SPE), Increase pH (basic) =

Answer 32

Negative

Question 33

(SPE), Decrease pH (acid) =

Answer 33

Positive

Question 34

T/F. In SPE, No charge at isoelectric point (Net charge: 0)

Answer 34

True

Question 35

The acidic and basic amino acids content of proteins determines its net charge

Answer 35

SPE

Question 36

SPE Buffer:

Answer 36

Barbital (Veronal) pH 8.6