兔磷酸激酶实验报告英文Word文件下载.docx

1、There are many ways of purifying creatine kinase,and the purification method used in this study is improved Kuby method.3. Measurement of creatine kinase activitiesThere are many ways of measuring the activities of creatine kinase, and we use pH-colorimetry in this study.pH-colorimetryWhen creatine

2、kinase is catalyze forward reaction, with the transfer of Phosphoryl from ATP to creatine, the equimolar H+ is produced, whose optimum pH is 7.59.0. Within this context, the generation rate of H + measured can be used as indicators of activity. In this study, thymol blue is used as a pH indicator. T

3、he activities of creatine kinase is measured by pH-colorimetry on spectrophotometer, which is monitoring changes in absorbance under the wavelength of 597nm. In the cuvette, H + produced by the enzyme-catalyzed reaction lower the pH value of solution. The color of substrate solution gradually change

4、 from deep purple red yellow green and lower the Absorbance value of A597. In the pH-colorimetry, the substrate solution requires to be new.4. Measurement of creatine kinase solution 280nm light absorptionMethod for protein measurementSensitivityTimePrincipleInterferenceCommentsSpectrophotometric（A2

5、80）Moderate50-1000 gRapid5-10 minAbsorption of 280nm light by aromatic residuesPurines, pyrimidines, nucleic acidsUseful for monitoring column eluents. Nucleic acid absorption can be corrected. Nondestructive to protein samples. Varies with proteins.II. Experimental1. Reagents and Equipments1） Reage

6、nts1. Preparation of crude extract:0.01M KCl, NH4Cl, 5M NH4OH, anhydrousethanol, pH8.5-9.0 2M MgSO4a. KCl：0.01mol/L,200ml. （0.15g KCl add water to 200ml）b. NH4OH：1.7mmol/L,1000ml.（take 0.12ml NH3H2O,add H2O to 1000ml） c. NH4Cl d. Anhydrous ethanole. MgSO4：2.0mol/L，pH8.5，20mlf. MgAC2：0.07mol/L，pH9.0，

7、100mlg. Tris-HCl：0.1mol/L，pH8.0，1000ml（Tris 12.1g, HCl 23ml, pH为8.0，add water to 1000ml）h. Salt2. Chromatographya. 48mmol/L creatine kinase solutionb. 0.1mol/L MgAC2c. 0.1 Thymol blue （100mg Thymol blue，dissolve with 20ml ethanol and 60ml ddwater）.d. 0.1mol/L pH9.0 Gly-NaOH buffere. ATPf. 0.1mol/L N

8、aOH g. 0.07M. MgAC2 pH9.0h. 0.01M Tris-HCl pH8.02） EquipmentsKnife, meat chopper, plastic basin, 1/100 balance, High-Speed diffuser, refrigerator;100ml beaker, 25ml beaker, 100ml measuring cylinder,250ml measuring cylinder;Pippet, burette, glass stick;0.5ml Ep tube, 1.5ml Ep tube;centrifugal machine

9、, 50ml centrifugal tube, stainless steel scoop, mortar;drinking paper, accurate pH test paper2. Measurement of enzymes activities:SPECORD200 Spectrophotometer, Pippete, 50ml beaker, burette.3. Measurement of protein solutions:SPECORD200 Spectrophotometer.2. Procedure1） Preparation of crude extract1.

10、 With a sharp knife, cut the muscle into cubes 4 to 5 cm wide from the ice-cold rabbit muscle.2. Quickly weigh about 50 g of the muscle cubes and suspend the cubes in 140 mL of 0.01 mol/L KCl at 0 C.3. Homogenize 140 mL of the suspension in a glass beaker with a blender （turn on the blender at high

11、speed for 20 seconds） and stir them with a glass stirring rod at 0 C for 15 minutes.4. Centrifuge the homogenate for 10 minutes at 10000 r/min at 0 C. 5. Carefully decant the supernatant and measure the volume of the supernatant （V1） （keep 0.6mL for protein assay at 20 C）. Discard the pellet.6. Add

12、ground ammonium chloride （NH4Cl） to make 0.1 mol/L solution and then adjust pH to 9.0 with 5 mol/L ammonium hydroxide （NH4OH）, keep stirring the solution at 0 C for 30 minutes.7. Add 1-fold V1 of cold 95% ethanol and stir the solution at 20 C for 30 minutes.8. Centrifuge the solution for 10 minutes

13、at 10000 r/min at -8 C. Discard the pellet and measure the volume of the supernatant （V2） （keep 0.6 mL for protein assay at 20 C）.9. Take the supernatant （V2） and add VA （mL） of 2 mol/L MgSO4 （pH 8.5） to a final concentration of 0.03 mol/L.10. Add 1-fold VA of cold 95% ethanol and stir the solution

14、at 0 C for 30 minutes.11. Centrifuge the solution for 10 minutes at 10000 r/min at -8 C. Pour off and discard the supernatant. 12. Suspend the pellet with 1/10 V1 0.07 mol/L MgAC2 （pH 9.0）. 13. Stir the suspension at 0 C for 30 minutes. Centrifuge the suspension for 10 minutes at 12000 r/min at 0 C.

15、 Pour off and discard the pellet. 14. Measure the volume of the supernatant （V3） （keep 0.6 mL for protein assay at 20 C）.15. Adjust the pH of the supernatant to 8.0 with 1 mol/L NaOH and add VB of cold 95% ethanol to a final concentration of 36% in an ice-salt bath. Calculate the VB using: （V3-the v

16、olume of MgAC2）x0.5+ VB/（V3+ VB）= 0.36.16. Stir the solution at 0 C for 30 minutes. Centrifuge for 10 minutes at 12000 r/min at -8 C. Pour off and discard the pellet. Measure the volume of the supernatant （V4） （keep 0.6 mL for protein assay at 20 C）.17. Add VC cold 95% ethanol to a final concentrati

17、on of 50% in an ice-salt bath. Calculate the VC using: （V4x0.36 + VC ）/ （V4 + VC ）= 0.6.18. Stir the solution at 0 C for 30 minutes. Centrifuge for 10 minutes at 12000 r/min at -8 C. Pour off and discard the supernatant. Suspend the pellet in about 5 mL of 0.01 mol/L Tris-HCl （pH 7.5）. Centrifuge fo

18、r 10 minutes at 12000 r/min at 0 C. Measure the volume of the supernatant （V5） （keep 3-4 mL for protein assay at 20 C）. 19. Store the V5 at 20 C overnight.2） Chromatography1. Q Sepharose High Performance column has been prepared for your use. Before starting a run, the column has to be equilibrated.

19、 This is done by pumping 5 column volumes of start buffer （buffer A: 0.01 mol/L Tris-HCl , pH 7.5） through the column. 2. Measure out 45 mL of V5 for application to the column, then wash the column with 2 column volumes of buffer A and elute the sample with buffer B （0.01 mol/L Tris-HCl, 0.3 mol/L N

20、aCl , pH 7.5） in a linear gradient, flow rate of the column is about 1 mL/min.3. Collect 1 mL fractions in test tubes in a fraction collector. Transfer the contents of every other fraction to a quartz cuvette and measure the absorbance at 280nm or use a UV monitor. Record the A280 values in your not

21、ebook. When you begin to detect protein eluting from the column （A280 0）, measure the A280 of every fraction.3） Measurement of Enzyme Activities1. The detection of the specific activity of the creatine kinase is based on the pH-colorimetry. It is rather sensitive for the advanced spectroscopy to mon

22、itor the alteration of the color of Thymol blue. 2. The way to calculate the specific activity is according to the following equation:1.3 is a converting coefficient. U stands for specific activity. VA stands for the volume of substrate （VA=1.0mL）. VB stands for the volume of enzyme solution （VB=0.0

23、1mL）. C is the concentration of enzyme solution, and it is determined by the absorbance of the solution at 280nm.3. Fill the specific activity of the creatine kinase in each fraction in the form bellow.Fraction Protein Concentration（mg/mL） Specific Activity Fold Purification Recovery （%） 4） SDS-PAGE

24、The samples obtained in each step of crude extraction and the final products from ion-exchange chromatography are loaded onto sodium dodecyl sulfate-poly-acrylamide gel electrophoresis （SDS-PAGE）. The stacking gel was made of 3% acrylamide, and the separating gel was made of 12.5% acrylamide. III. Experimental record1. The volume in each purification stageChart 1. Volume of the protein.V1V2V3V4V546.8 ml83.1 ml4.98 ml5