Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

!Experiment 1

628 views

Published on

  • Be the first to comment

  • Be the first to like this

!Experiment 1

  1. 1. E xperiment 1 Preparation of Elastic Protease from Pancreas and Analysis of Enzyme Activity Department of Biological Pharmacy, China Pharmaceutical University, 2011
  2. 2. CONTENTS 1 Purposes 2 Part I: Preparation of Elastic Protease 3 Part II: Analysis of Enzyme Activity
  3. 3. Purposes 1 Learn the principle of preparation of elastic protease from pancreas. 2 Master the analysis method of enzyme activity.
  4. 4. Part I Preparation of Elastic Protease
  5. 5. Procedures I Frozen Pancreas Frozen Pancreas 75 g 75 g (1) Activate the enzyme at 24℃ for 24h (2) remove the fat, cut into pieces (3) add 50ml HAc-buff ,mashed Pancreatic Pancreatic Plasma Plasma (1) add 250 mL HAc-buff, adjust pH to 4.5 (2) 25℃ stir for 1 hours, (3) Filter Continued on Continued on Filtrate Filtrate next page … next page …
  6. 6. Procedures II Continued from Continued from Filtrate Filtrate previous page … previous page … (1) add 40 g resin for adsorption (2) 25℃ stir for 1.5 h (3) wash with water Resin Resin (to absorb protein) (to absorb protein) (1) add 50 ml 1 M NH4Cl-buff, adjust pH to 9.0 (2) stir for 1 h (3) adjust pH to 5.2~6.0 (4) filter Continued on Continued on Eluate Eluate next page … next page …
  7. 7. Procedures III Continued from Continued from Eluate Eluate previous page … previous page … Add 3-time-volumn cold acetone Precipitation Precipitation (1) wash twice with acetone (2) wash once with ether (3) vacuum drying Elastase powder Elastase powder End Product Obtained
  8. 8. Part II Analysis of Enzyme Activity
  9. 9. Definition Definition of Enzyme Activity The amount of enzyme hydrolyzing 1.0 mg Congo red elastin within 20 min at pH 8.8 and 37 ℃ is defined as an activity unit.
  10. 10. Preparation of Reference Substance I 1 30 mg Congo red elastin (taken accurately) 4 Shake the bottle gently 100 mL until all substrates volumetric flask dissolves ( about 60 min) 2 20 mL standard elastic enzyme solution (must be sufficient) 3 Keep the flask at 37 ℃ in a water incubator
  11. 11. Preparation of Reference Substance II 5 50 mL phosphate buffer (pH 6.0) 4 Shake the bottle gently 100 mL until all substrates volumetric flask dissolves ( about 60 min) 6 add boric acid buff to the scale 3 Keep the flask at 37 ℃ in a water incubator
  12. 12. Sample Preparation 1 2 5 mL pH 8.8 boric acid buff (add a small amount at first) 5 mg enzyme sample (taken accurately) mortar 3 grind until everything dessolves 1 mL boric acid buff 4 Dilute enzyme solution with boric acid buff to 2 ~ 3 unit/mL
  13. 13. Standard Curve 1 2 Take series amounts of reference substance solution, adding mixture of boric acid buffer and phosphate acid buffer (1:1), as indicated in the table below. Measure the absorption at 495nm, using the absorption of tube 0 as control. Plot a standard curve. Tube No 0 1 2 3 4 5 Reference substance (mL) 0 2.0 4.0 6.0 8.0 10.0 10.0 8.0 6.0 4.0 2.0 0 Mixture of buffer (mL) A495 value
  14. 14. Sample Preparation Take 3 tubes to operate according to the table below: Tube No. Congo red elastin (mg) pH 8.8 boric acid buff (mL) 0 1 2 1.2 3 3 5 4 4 Await measuring enzyme liquid 1 1 (mL) Hydrolyze the mixure at 37℃ for 20 min (The intermittence is stirred 20 or more times) pH 6.6 phosphoric acid buff (mL) 5 5 5 Take the supernatant after centrifugation (3000 rpm × 10 min) A495 value
  15. 15. Caculation 1 Take the average absorption and check the corresponding enzyme activity according to the standard curve. 2 Calculate the relative enzyme activity (enzyme activity/enzyme amount) by taking into account the dilution times. 3 Calculate the total enzyme recovery yield.
  16. 16. The End It’s time for your practice. EVERYBODY GO GO GO! PPT 设计&制作: 中国药科大学 · 生物制药教研室 2011.9

×