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《 生化大实验 》 PowerPoint PPT Presentation


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《 生化大实验 》. 实验课程简介. 生化大实验是一门综合性的实验课程,教学的目的在于通过对生命物质的分离制备,纯化,分析等综合性实验,在已掌握的基础生物化学理论知识和生化实验常用方法的基本原理、基本操作技术(如滴定、比色、层析、电泳等)的基础上,训练学生的综合实践动手能力,掌握蛋白质、酶、核酸等重要物质的分离、纯化等的测定技术。. 目 录. 实验一 蛋白质含量的测定 实验二 离子交换法血清纯化 IgG 实验三 离子交换层析法分离血清白蛋白 实验四 SDS- 聚丙烯酰胺凝胶电泳测定分子量 实验五 等电聚焦法测定样品的等电点

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《 生化大实验 》

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6051133


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IgG

SDS-

PCR


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--


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G-250--465nm595nm595 nm 2min1h-10-100g1-10g


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1. 0.1mg/ml0.2g0.9%NaCl 2000ml

2. 0.9%NaCl

3. 100mgG25050ml 5%100ml85%1000ml

4. 0.9%NaCl200

5.


6 0 5ml 2 1 0ml 2 5ml 1 7 8 1 5cm 15cm 8 9 100ml 1 10 11 1

6. 0.5ml21.0ml2 5ml17. 8. 1.5cm15cm (8)9. 100ml(1)10. 11. (1)


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7

A595


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2. 1.0ml4.0ml3min595nm 15-20min2-


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1

1

260

330

2


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1.

2.

280nm


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2260280nm 0.11.0mg/mL


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1. 10ml52. 0.9%NaCl0.25g250ml3. 1.0-2.5mg/ml 4. 0.9%NaCl5. 1.5cm15cm96. 1ml32ml25ml2


1 1 8 2 5 1cm 280nm a280 a280

1 182-51cm 280nmA280A280

1-2 ---


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2 1ml3mlA2802. 280nm260nm mg/mL=1.45A280-0.74A260 (Cmg/ml,A280A260280nm260nm)


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A2800.75280nm 1.2.


Folin

Folin-

Folin-

Folin-lowry--750nm660nm


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1. Folin-A20g4g1000ml10.5g1g Na3C6H5O7100ml2

50ml11ml2A,

2. Folin-B100g25g100g 50ml80%100ml1500ml10h150g50ml15min100ml1mol/L


3 200 g ml 4 721 5 0 5ml 1 1ml 2 5ml 1 6 1 5cmx15cm8 7 1

3200g/ ml472150.5ml11ml25ml161.5cmX15cm871


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6:


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A10minFolin-B 3.0ml10min0.2ml30min660nm2 1ml660nm 1Folin-2Folin-


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C u2540nm


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1. 2.0mg/mL2. 10ml63. 1.5cm15cm84. 0.175gCuSO4 5H2O15ml100ml30ml 30ml20ml1-2h100ml300ml10%1000ml


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5. 1ml2ml5ml6. 721 1. 66 701234561-63.0ml03.0ml 2.0 ml540nm30min


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2. 3.0ml2.0ml540nm 1. 2.


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IgG

DEAE-Sephadex A-50 - - A-50 NaOH Cl - OH - pH6.85 7.5 pH 7.2 7.4 DEAE-Sephadex A-50 IgG


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1. DEAE-Sephadex A-50

2. 0.5mol/L HCl NaOH

3. 0.1mol/L pH7.4 PBS

4. 0.1mol/L Tris-HCl(pH7.4)

5. 0.02 NaN 3

6. PEG

7.

8.

9. 1cm20cm

10.


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1 DEAE-Sephadex A-50 DEAE-Sephadex A-50 A-50 5g 500ml 1h A-50 0.5mol/L NaOH 15ml 0.5mol/L NaOH 30min 2 pH 0.5mol/L HCl 0.5mol/L NaOH A-50 0.1mol/L pH7.4 PBS


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2

1

2 0.1mol/L Tris-HCl(pH7.4) 1/4 A-50 A-50 2 3cm 1ml/min A-50

3 A-50


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3 4 /min 2 pH PH

4 0.5ml 2% 100mg 2 3 4 /min.


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5 4ml. 10 15 6 751 A 260 A 280 7 PEG Mw6000 0.02 NaN 3 4 -40 8 A-50 2mol/L NaCl A 280 <0.02 A-50 4 2 50


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pH


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pH


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1. 20%

2. DEAE32cellulose

3. PBS0.0175M pH6.70.2M Na2HPO4 3.5mL0.2M NaH2PO4 56.5mlpH6.787.5ml1000mL

4. 0. 1N HCl0.1N NaOH

5. Nessler

6. 1cm20cm

7.


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1. DEAE32(1)DEAE321.5gDEAE3210~150.1N NaOH300ml(2)pH.8.0(3)0.1N HCl30minpH.6.0


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(4)PBS0.0175M pH6.73hr(5)PBS0.0175M pH6.71.5 2. 1/3PBS 0.0175M pH6.7,DEAE32cellulose 3. PBSpH6.7


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4. -3-PBS0.0175M pH6.712-24hrPBSpH6.75. IgGPBS0.0175M pH6.70.3ml/minEppendorf1ml30%PEG-


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6. (1)7. 1M NaCl0.02M NH4AcpH6.51%


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8. , PBS0.0175M pH6.724hrPBSpH6.7 0.06 mol/L NH4Ac pH6.5 30 ml0.3 mol/L NH4Ac pH6.520%


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0.3 mol/L NH4Ac 0.3 mol/L NH4Ac 4 ml31030%PEGDEAEcellulose20 ml 1.5 mol/L NaCl 0.3 mol/L NH4Ac 40 ml 0.02 mol/L NH4AcpH6.51%


1 2 3

1. 2. 3.


Sds page

SDS-PAGE

1.SDS-PAGE

2.

3.SDS-PAGE


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1 2 3 1 igg 2

1. 2 31. IgG2.


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1. Acr30gBis0.8gddH2O100ml2. 0.05M pH8.0 Tris-HCl0.61 gTris 50ml ddH2O3ml 1M HClpHpH8.0ddH2O100ml3. Tris 36.3g1M HCl48.0mlddH2O100mlpH8.9


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4. 3 g Tris14.4 g1g SDSddH2O1000ml5. 1.0ml0.1ml100mgSDS2mg2ml Tris-HCl pH8.0 ddH2O10ml6. AP1gAP10ml ddH2O7. TEMED


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8. 1.25 g R-250500ml9. 75mL50mL875ml10. 11. Tris 5.98g1M HCl 48.0mlddH2O100mlpH6.712. 50%454mL46ml


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1. 1%1g100ml/2. 2.5mlTrispH8.93.3ml4.2ml50l-10%SDS50l AP10l TEMED; 3. 1~2cm30min


1 25mltris ph6 7 0 85ml 2 95ml 20 l 10 sds 25 l ap 10 l temed

1.25mlTrispH6.70.85ml2.95ml20l-10%SDS25l AP10l TEMED


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4. 5. 10-20l10-20l1003~5min6. 10l7. 100V120V


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1cm8. 30min9. 30min


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10. 3711. 12.


13 1 2

13 1= / / 2= /


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14


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1 .0.5g/kg,,,,,,. 2,,,,,,. 31mg/mL1mg/mL1mg/mL1mg/mL


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1. SDS-PAGE,,,? 2. ? 3. SDS-PAGE,TEMEDAP,? 4. ?


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1.

2.


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pHpHpHpHpHpHpHpHpH


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1. 2()1. IgG2. 1. AmpholinepH 3~1040%2. AcrAcr 30g Bis1.0g100ml4


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3. TEMED410% AP50.2%(V/V)(85%29.4ml500ml)60.5%(V/V)(NaOH10g500ml)710%(W/V)8R250 2.0 g50% 1000ml93ml7.0ml9551


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7.5 %

7.0ml

30%2.5ml

Ampholine0.3ml

0.1ml

AP10%0.05ml

TEMED0.01ml


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AP50ml10min0.3ml Ampholine0.1ml IgG0.01ml TEMEDSDS-PAGE5mm3mm20~30minPI=6.7CPI=10.25PI=3.75


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2. 0.2%0.5%/NaOH160V30min3.


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4. 30min 310minAmpholine30min3~5


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5. pH0.5cm1mlpHpHpH


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6 1pHmmpHPHpH5mmpHpH5mmpHpH2.5mmPH522.5mmnPH5n2.5 mm


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2 LpLp=lpl1 / l2 lpl1l2 LppH


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1. pHIEF-PAGESephadexG-252. R-25050-150g1g0.5-3 mg/mL10-30l


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3. pHpH2 4. 810cmpH 5. 2Triton X100CHAPSZwittergent314


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1

2.


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DNADNA

pH12.0-12.5

DNADNAph4.8pHDNADNARNA-SDS


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1.

2.

3.

4.


5 6 7 8 9 10 rna 11 12

5. 6. 7. 8. 9. 10. RNA11. 12.


13 14 15 8 16 17 18 puc19 20

13. 14. 15. 8-16. 1718. puC1920.


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1. 50mmol/l 25mmol/l 10mmol/l2. 0.4mol/l 2%SDS3.


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5mol/L 60ml 11.5ml 28.5ml 4. TE10mmol/L Tris.Hcl(PH8.0) 1mmol/L Edta(PH8.0) 5. 70%6. RNA RNA10mmol/L Tris.Hcl15mmpl/L10mg/mL10015-207. (6x)40%0.25%8.


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1. 2LBpuC1937

2. 1.54000r/min2

3.

4. 100ul10


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5200ul56. 150Ul3157. 12000r/min158. 1112000r/min59. 25-1012000r/min5


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10.1ml70%DNA11.20ulTE20ul/mlRNADNA-20DNA DNADNA


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PCR

1. PCR

2.

3. PCR


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polymerase chain reaction, PCRDNADNADNA2n

1. DNA94

2. 5060 DNA


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3. 72 DNADNA4dNTP,53DNA 33DNA2530DNA106109 PCRDNAdNTP MgCl2DNATaq DNA


1 pcr 2 3 4 5

1. PCR2. 3. 4. 5.


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1. DNA2. primer1 primer21. PCRTaq DNA4dNTP2.5 mmol/L2. 53. 50TBETris 242 g57.1 mL0.5mol/L EDTA(pH 8.0)100ml1000ml


1 0 2ml taqdna 50 l

1. 0.2mLTaqDNA50L


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2. PCR 94 5min 94 1min 54 1min 72 1min 30-35 72 10min 20 1min


3 pcr 2 3 l 1 l dna eb 15min

3. PCR 23 L1L DNAEB15min


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1. PCRPCR2. Mg2+TaqDNAMg2+3.


1 pcr 2 tm 3

1.PCR2.Tm3.


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ELISA60ELISA3


Elisa

ELISA, ----


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1. (Bio550) (96)2. (1)HRPIg G(1:1 000)IgG1:256(2)0.05mol/L pH9.6, 4,Na2CO3 0.15g, NaHCO3 0.293g,100 mL


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(3)0.01mol/LpH7.4 PBS--Tween-20, NaCl 8g, KH2PO4 0.2g, Na2HPO412H2O 2.9g, Tween200.5m1000m(4)0.1%0.01mol/L PBS pH7.4(5)()Na2HPO412H2O 2.9g,0.51g100mL, 40mgOPD40L30%H2O2(6)2mol/LH2SO4500mL98%76mL


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1. IgG10mg/mL0.1g/mL100L0%4834100%28+2

2. IgGIgG10mg/mLIgG4g/mL0.325g/mL200L32200L4IgG100%4


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3. 200L1min43745min4. IgG100L4100%372h45. HRPIg G100LHRP-IgG371h


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6. OPD4100L3720min50L7. 492nm8. Ig(IgG)B/BoCIgGBIgG0%;Bo1000%


B b0 igg

B/B0IgG


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1. 2. ELISA3.


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