LAB: EXP 2 (Quali & quanti tests for proteins)

Question 1

Biuret:
Purpose, Reagent, (+) Result, Principle

Answer 1

• Purpose: Used to detect the presence of peptide bonds
• Reagents: NaOH & CuSO4
• (+) result: pink to violet to blue coloration
• Principle: Complexation of Cu2+ with a peptide bond

Question 2

Ninhydrin:
Purpose, Reagent, (+) Result, Principle

Answer 2

• Purpose: detects free α-amino acids & amines
• Reagents: 1,2,3-indanetrione monohydrate/ triketohydrindene & EtOH
• (+) result: blue to blue-purple color or yellow-orange product (Proline)
• Principle: oxidative decarboxylation & deamination followed by condensation

Question 3

Xanthoproteic:
Purpose, Reagent, (+) Result, Principle

Answer 3

• Purpose: detects aromatic amino acids (Y, W & F)
• Reagents: conc. HNO3 & conc. NaOH
• (+) result: yellow sol’n (heating); orange sol’n (x’ss NaOH)
• Principle: nitration of aromatic ring

Question 4

Millon’s:
Purpose, Reagent, (+) Result, Principle

Answer 4

• Purpose: phenolic group in Tyrosine
• Reagents: HgSO4 in H2SO4
• (+) result: old rose/ flesh/ purple-red ppt.
• Principle: complexation (mercuration & nitration or nitrosation/complexation of nitrohydroxyphenyl derivatives with Hg2+)

Question 5

Hopkin’s cole:
Purpose, Reagent, (+) Result, Principle

Answer 5

• Purpose: indole group in Tryptophan (Trp)
• Reagents: glyoxylic acid & conc. H2SO4
• (+) result: pink to violet interface
• Principle: reduction of oxalate to glyoxylate and acid-catalyzed

Question 6

Sakaguchi:
Purpose, Reagent, (+) Result, Principle

Answer 6

• Purpose: guanido group of Arginine
• Reagents: α-naphthol, NaOBr, NaOH & Urea
• (+) result: red to red-orange color
• Principle: base-catalyzed condensation of α-naphthol with the guanido group of arginine

Question 7

Fohl’s/Lead:
Purpose, Reagent, (+) Result, Principle

Answer 7

Purpose: S-containing amino acids ( M & C )
• Reagents: Lead (II) acetate & NaOH
• (+) result: brown or black ppt.
• Principle: degradation and substitution reaction to form
PbS

Question 8

Pauly’s/Diazo:
Purpose, Reagent, (+) Result, Principle

Answer 8

• Purpose: detects the presence of histidine and tyrosine
  • Reagents: sulfanilic acid, NaNO2 & Na2CO3
  • (+) result: red color
  • Principle:
  • diazotized sulfanilic acid couple with amino phenol to form
  a color red azo compound in cold conditions

Question 9

Nitroprusside:
Purpose, Reagent, (+) Result, Principle

Answer 9

• Purpose: presence of –SH group (cysteine)
• Reagents: Na2Fe(CN)5NO in dilute NH3
• (+) result: red coloration
• Principle: complexation

Question 10

Amide test:
Purpose, Reagent, (+) Result, Principle

Answer 10

• Purpose: It detects 1o, 2o, 3o amides and nitriles
• Reagents: NaOH
• (+) result: red litmus paper 🡪 blue
• Principle: Base-catalyzed Hydrolysis

Question 11

Principle of chromatography

Answer 11

Based on affinity

Question 12

Stationary phase in chromatography

Answer 12

Silica gel

Question 13

Moving/mobile phase in chromatography

Answer 13

ButOH:HOAc:H2O

Question 14

Ratio of ButOH:HOAc:H2O

Answer 14

4:1:5

Question 15

Chromatography is visualized using ____

Answer 15

Ninhydrin

Question 16

Used to determine the amino acid composition of a given protein solution

Answer 16

Chromatography

Question 17

Enumerate the stages in TLC

Answer 17

• Sample/Std. Application
• Development
• Visualization
• Evaluation
• Documentation

Question 18

A simple colorimetric and one of the most commonly used assay for total protein concentration

Answer 18

Bradford assay

Question 19

Principle of bradford assay

Answer 19

proportional binding of Coomassie dye to proteins due to hydrophobic and electrostatic interactions

Question 20

λ max of dye in bradford assay

Answer 20

465-595 nm

Question 21

The Coomassie Brilliant Blue G-250 (dye), reacts primarily to _____ and _____

Answer 21

basic (especially arginine), aromatic amino acids

Question 22

Bradford reagent is a dye dissolved in _____ and _____

Answer 22

ethanol, phosphoric acid