Protocolo

M.A. Navarrete del Toro

Lab. Bioquímica CIBNOR

Methods for Laboratory of Biochemistry
CONTENTS Anatomy of Penaeus Recovery of enzyme crude extracts Protein quantification Ultraviolet absorption Bradford method Lowry method Introduction to Proteins: Protein hydrolysis Proteolytic activity assay Inhibition of proteolytic activity by specific inhibitors Inhibition of proteolytic activityassay Trypsin and chymotrypsin amidase activity Trypsin activity assay Chymotrypsin activity assay Acid proteolytic activity assay 2 3 5 6 8 12 15 16 18 20 21 22 22 24 25 26 30 33 35 40 41 42 43 44 46 49 50 1

Protein electrophoresis
Principles of electrophoresis SDS- Polyacrylamide Gel Electrophoresis (SDS-PAGE) Working with (SDS-PAGE) Fixing the gels Preparing the sample Loading sample Runningthe gel Staining and destaining Molecular weight evaluation for SDS-PAGE Evaluating the presence of alkaline enzyme activity by Substrate-SDS-PAGE Evaluating the presence of acid enzyme by SubstrateNeutral PAGE

Protein hydrolysis using the pH-stat technique

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M.A. Navarrete del Toro

Lab.Bioquímica CIBNOR

Anatomy of Penaeus
External anatomy

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M.A. Navarrete del Toro

Lab. Bioquímica CIBNOR

Internal anatomy 1.

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M.A. Navarrete del Toro Internal anatomy2.

Lab. Bioquímica CIBNOR

Mouth

Hepatopancreas Anus Foregut Midgut

Digestive system, ventral view Hepatopancreas

Foregut

Midgut Digestive System, dorsal view

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M.A. Navarrete del Toro

Lab. Bioquímica CIBNOR

Extraction of the whole digestive system organs.

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M.A. Navarrete del Toro

Lab. Bioquímica CIBNOR

Extraction of hepatopancreas
The hepatopancreas is the digestive gland, located inside the cephalotorax. 1. Separate the head from the shrimp by twisting and pulling the head out from the abdomen. 2. Using a small scissors, cut off over thelateral side of the head until the hepatopancreas is exposed. The hepatopancreas looks like a white chickpea 3. Take the hepatopancreas out from the cephalotorax and separate it from the stomach and the midgut.

Hepatopancreas

4.

Put the hepatopancreas in a weighted glass beaker, keeping the beaker on ice

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M.A. Navarrete del Toro

Lab. Bioquímica CIBNOR

Preparation of the enzyme crude extract
1. When all the hepatopancreas are collected, weight them together, and add cold distilled water in a ratio of 1:3 w/v. 2. Homogenize the hepatopancreas using a hand blender or a tissue homogenizer at 10,000 rpm for 15 seconds two times. 3. Centrifuge the homogenate at 10000x g for 30 min at 5 °C 4. The supernatant called the “enzyme crude extract” is maintained at 4 °C. 5. Evaluate the concentration of the soluble protein and protease activity. 6. The enzyme extract must be stored at -20 °C, in small aliquots, or freezedry and store the powder at -20 °C. 7. Evaluate the protein content and protease activity to the extract before and after freezing or freeze-drying,to know if the storage methods affect the activity of the enzymes.

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M.A. Navarrete del Toro

Lab. Bioquímica CIBNOR

Quantification of protein concentration.
Methods used for evaluation of protein concentration yield different results because they react differently with the...