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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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IgG

SDS-

PCR


--


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


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

2. 0.9%NaCl

3. 100mgG25050ml 5%100ml85%1000ml

4. 0.9%NaCl200

5.


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


7

A595


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


1

1

260

330

2


1.

2.

280nm


2260280nm 0.11.0mg/mL


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


1 182-51cm 280nmA280A280

1-2 ---


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


A2800.75280nm 1.2.


Folin-

Folin-

Folin-lowry--750nm660nm


1. Folin-A20g4g1000ml10.5g1g Na3C6H5O7100ml2

50ml11ml2A,

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


3200g/ ml472150.5ml11ml25ml161.5cmX15cm871


6:


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


C u2540nm


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


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


2. 3.0ml2.0ml540nm 1. 2.


IgG

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


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.


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


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


3 4 /min 2 pH PH

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


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



pH


pH


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.


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


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


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


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


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


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.


SDS-PAGE

1.SDS-PAGE

2.

3.SDS-PAGE



1. 2 31. IgG2.


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


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


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


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


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



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


1cm8. 30min9. 30min


10. 3711. 12.


13 1= / / 2= /


14


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


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


1.

2.


pHpHpHpHpHpHpHpHpH


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


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


7.5 %

7.0ml

30%2.5ml

Ampholine0.3ml

0.1ml

AP10%0.05ml

TEMED0.01ml


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


2. 0.2%0.5%/NaOH160V30min3.


4. 30min 310minAmpholine30min3~5


5. pH0.5cm1mlpHpHpH


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


2 LpLp=lpl1 / l2 lpl1l2 LppH


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


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


1

2.



DNADNA

pH12.0-12.5

DNADNAph4.8pHDNADNARNA-SDS


1.

2.

3.

4.


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


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


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


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.


1. 2LBpuC1937

2. 1.54000r/min2

3.

4. 100ul10


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


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


PCR

1. PCR

2.

3. PCR


polymerase chain reaction, PCRDNADNADNA2n

1. DNA94

2. 5060 DNA


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


1. PCR2. 3. 4. 5.


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


1. 0.2mLTaqDNA50L


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


3. PCR 23 L1L DNAEB15min


1. PCRPCR2. Mg2+TaqDNAMg2+3.


1.PCR2.Tm3.



ELISA60ELISA3


ELISA, ----


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


(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


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

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


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


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


B/B0IgG


1. 2. ELISA3.


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