复旦大学：《有机化学实验 Organic chemistry lab》课程教学资源（实验讲义）The isolation of casein（from milk）

E Organic chemistry experiment The isolation of casein(from milk) Experimental center of chemical education Fudan University

The isolation of casein (from milk) Organic chemistry experiment Experimental center of chemical education Fudan University

Target 1. Comprehend the concept of pI 2. Isolate the protein by adjusting the ph 3. Identify the protein through biuret test

Target 1.Comprehend the concept of pI 2.Isolate the protein by adjusting the pH 3.Identify the protein through biuret test

NIV Background C asein NH/HO GOLD STANDARD CASEIN Ho.∠NH 4.8

Background pI=4.8 Casein

迫 Experimental principle 1. Casein is a kind of complex phosphoprotein, and due to its nature of being amphoteric compound, the acid-base property of the solution will obviously affect the charge on protein. When the ph of milk adjusted close to the pl of casein (pl=4.8), the protein becomes neutral. Since its lowest solubility, sediment will form, which could be separated through centrifugation NH NH2 H salt high solubility low solubility salt( high solubility)●

1.Casein is a kind of complex phosphoprotein, and due to its nature of being amphoteric compound, the acid-base property of the solution will obviously affect the charge on protein. When the pH of milk adjusted close to the pI of casein (pI=4.8), the protein becomes neutral. Since its lowest solubility, sediment will form, which could be separated through centrifugation. salt （ high solubility ） low solubility salt （ high solubility ） Experimental principle

NIV 迫 Experimental principle 2. Biuret test: The compound with polypeptide skeleton can react with Cu2+ under basic condition which will form purple complex A"9人° Protein or polypeptide urple complex

2. Biuret test ：The compound with polypeptide skeleton can react with Cu2+ under basic condition, which will form purple complex. Protein or polypeptide Purple complex Experimental principle

迫套 Reagents Reduced-fat milk Dilute acetic acid (1: 9) Ethyl alcohol Diethyl ether 1% CuSO 0. 4MNaoh in saline

Reduced-fat milk Dilute acetic acid (1:9) Ethyl alcohol Diethyl ether 1% CuSO4(aq) 0.4 M NaOH in saline Reagents

Reagents M p b p Diethyl ether 74.12 116 35 0.713 EtoH 46.07 114 78 0.789

Diethyl ether 74.12 -116 35 0.713 EtOH 46.07 -114 78 0.789 M.w. m.p. b.p. d. Reagents

The isolation of casein 1. 50 mL Reduced-fat milk. 100 ml beaker 40 oC Dilute acetic acid (1: 9)2 mL 2. Cool down for 10 min, 3000 r/min 15 min centrifugation 3. Separate the sediment, mixed with 95%o etoh 20 mL suction filtration 4. 10 mL EtOH: Et2O=1: I wash twice, 5 mL Et,O wash suction filtration 5. Dry and weig h 6. Solute 0.5 g product in 5 mL 0.4M NaOH, biuret test

The isolation of casein 1. 50 mL Reduced-fat milk, 100 mL beaker 40 oC Dilute acetic acid ( 1:9 ) 2 mL 2. Cool down for 10 min， 3000 r/min 15 min centrifugation 3. Separate the sediment, mixed with 95% EtOH 20 mL , suction filtration 4. 10 mL EtOH：Et2O = 1:1 wash twice， 5 mL Et2O wash suction filtration 5. Dry and weigh 6. Solute 0.5 g product in 5 mL 0.4 M NaOH, biuret test

Attention 1. The milk could not be long-placed or the lactose will transform to lactic acid which may affect the isolation 2. Excess acetic acid is not allowed. since the acetic acid will accelerate the hydrolysis of lactose

1. The milk could not be long-placed, or the lactose will transform to lactic acid, which may affect the isolation. 2. Excess acetic acid is not allowed, since the acetic acid will accelerate the hydrolysis of lactose. Attention