TEACHING PLAN
for biochemistry experiments
Course：Biochemistry
Students： oversea undergraduates
Periods：44 lessons
一、Aims of the practicals
Progress in science is achieved through observation and experiment. Biochemistry is an experimental
science that advances from well-thought out investigations in the laboratory. No serious student should neglect
the opportunities which this course provides to appreciate this fact. Your course includes experiments for you
to gain some insight into how laboratory investigations are carried out and how data are processed and
interpreted. To obtain useful results an experiment should be designed to answer a definite question and the
detailed planning should be sufficiently rigorous to exclude adventitious errors. The course gives you the
opportunity to plan some experiments for yourselves. You should benefit from the practicals in two ways:
(i) You will learn a variety of experimental techniques, all of which are currently used in biochemical
research. The practicals have been designed to complement the lectures and fit in with their sequence as far as
possible. The hands-on experience should link to the mental framework provided by the lectures, and give you
a deeper understanding and more realistic perspective of the topics discussed.
(ii) You will learn to handle experimental data effectively, and to extract the maximum information
content without falling into the trap of over-interpretation.
二、The contents of biochemistry experiments:
Experiment 1
ⅰ Basic operation for biochemistry experiment
ⅱAssay of protein concentration（Coomassie Brilliant Blue G-250）
【Aims】
1．To learn how to wash glass utensils
2．To learn how to use pipettes
3．To learn how to use 721 spectrophotometer
4．To learn how to assay protein concentration with Coomassie Brilliant blue G-250
【Periods】
3 classes
【Contents】
1．To emphasize the laboratory rule
2．How to write experience report
3．To introduce how to wash glass utensils
4．To introduce how to use 722 spectrophotometer
5．To introduce the principle of assay of protein concentration with Coomassie Brilliant Blue G-250 and
the method of making a standard curve.
6．To assay blood serum specimen
【Methods】
1．To give a demonstration of using the pipettes and 722 spectrophotometer.
2．To assay serum protein concentration with standard curve.
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Experiment 2
ⅰ Electrophoresis of serum protein (CAME)
ⅱAssay of protein PI
【Aims】
1．To understand and master the principle of CAME
2．To learn how to analyze relative concentration of various
protein in serum
3．To strengthen the comprehension of the protein ionization and pI
4．To learn how to assay protein pI
【Periods】
3 classes
【Contents】
1．To review pI and introduce the principle of CAME
2．Preparation before Electrophoresis
3．Electrophoresis
4．Dyeing and rinsing
5．To make a series pH buffers
6．To observe protein precipitation in the divers pH buffer
【Methods】
1．To separate serum protein with CAME.
2．To assay protein pI with precipitation method.
Experiment 3
ⅰ Gel filtration ( Sephadex G25 )
ⅱDiscussion: physicochemical property and their application, protein and diseases
【Aims】
1．To understand and master the principle of Gel filtration
2．To give a deeper understanding of physicochemical properties of proteins and the relationship between
protein and diseases
【Periods】
3 classes
【Contents】
1．To introduce the principle of Gel filtration
2．Loading the column
3．Loading the sample
4．Eluting the column and collecting
5．To draw the curve of elution
6．To discuss the physicochemical properties of proteins and the relationship between protein and diseases
【Methods】
1．To separate the mixture solution of Potassium dichromate and Dextran blue with gel filtration.
2．To organize the discussion.
Experiment 4
Assay of Km Value of Alkaline Phosphatase
【Aims】
1. To understand the effect of the changes of substrate concentrations on the velocity of enzyme-catalyzed
reaction.
2. To learn the principle and methods of determining Km value and its role at enzyme kinetics.
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【Periods】
3 classes
【Contents】
1．To introduce the principle of assay of Km value
2．Assay of Km
【Methods】
To assay Km with Lineweaver-Burk double-reciprocal plot.
Experiment 5 Assay of Activity of Alkaline Phosphatase
【Aims】
1．To master the basic method of the assay of the AKP activity.
2．To strengthen the comprehension of enzyme activity and its inhibition.
3．To be familiar in the meaning of the activity unit, total activity , ratio activity and how to do the
calculation of AKP activity.
【Periods】
3 classes
【Contents】
1．To introduce the principle of assay of alkaline phosphatase activity
2．Make the standard curve
3．Assay of AKP activity
【Methods】
Assay of AKP activity with standard curve
Experiment 6
ⅰ LDH and Its Coenzyme
ⅱ Function and Inhibition of Cytochrome
【Aims】
1．To understand the actions of lactate dehydrogenase ( LDH ) and its coenzyme.
2．To have an appreciation of the extract method about the raw lactate dehydrogenase apoenzyme and its
coenzyme.
3．To understand the actions of cytochrome system and the inhibition of cyanide on cytochrome oxidase.
【Periods】
3 classes
【Contents】
1．The preparation of muscle and heart homogenate, respectively.
2．The preparation of raw extract of NAD+:
3．The preparing of apoenzyme of lactate dehydrogenase
4．Observe the actions of apoenzyme of lactate dehydrogenase and its coenzyme
5．Observe the function and inhibition of cytochrome
【Methods】
1．The actions of apoenzyme of lactate dehydrogenase and its coenzyme is observed through methylene
blue reduction.
2．The function and inhibition of cytochrome is observed through p-diamino benzene oxidation.
Experiment 7
Design of Experiment about the Best Conditions for Amylase Activity
【Aims】
1．To get the thread how to design a project about experiment research by the design of experiment and
should be able to put it in practice.
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2．To get deeper insight into some methods of enzyme kinetic research, for example, how does the change
of pH affect the reactions catalyzed by enzyme.
【Periods】
3 classes
【Contents】
1. To prepare saliva amylase and dilute it
2. To make up the diverse pH solutions
3. Measure the activity of saliva amylase in different pH solution.
4. Draw the graph and find the best pH for the saliva amylase.
【Methods】
To observe the activity of saliva amylase in different pH solution by the method of starch hydrolyzation..
Experiment 8
ⅰ Adrenalin and Insulin Influence for Glucose Concentration in Serum
ⅱ Determination of Glucose in Urine
【Aims】
1．To master the influence of adrenalin and insulin on glucose concentration in serum.
2．To know the method of determination of glucose in urine and normal value of Glucose Concentration In
Serum.
【Periods】
3 classes
【Contents】
1. To introduce the principle of assay of glucose in serum and urine respectively.
2. To obtain the serum from the rabbit before and after inject the adrenalin or insulin
3. To assay concentration of glucose in serum
4. To detect glucose in urine
【Methods】
1．To assay concentration of glucose with the method of glucose oxidase-peroxidase.
2．To detect glucose in urine with the method of copper ion reduction.
Experiment 9
ⅰ Assay of Total Cholesterol Concentration in Serum
ⅱ Assay of Urea Concentration in Serum
【Aims】
1. To grasp the principle and methods of assay of cholesterol concentration in serum using cholesterol
oxidase-peroxidase.
2. To learn about normal value of cholesterol concentration in serum and its clinical significance.
3. To have an appreciate understand of the assay method of urea in serum and its clinic significances.
4. To review the mechanism of the producing of urea in liver and its significances.
【Periods】
3 classes
【Contents】
1. To introduce the principle of assay of concentration of cholesterol and urea in serum respectively
2. To assay concentration of cholesterol in serum.
3. To assay concentration of urea in serum.
【Methods】
1．To assay concentration of cholesterol with the method of cholesterol oxidase -peroxidase.
2．To assay concentration of urea with the method of diacetyl monohydroxime.
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Experiment 10
ⅰ Assay of ALT Activity in Serum
ⅱ Determination of Ketone Bodies in Urine
ⅲ
Determination of Proteins in Urine
【Aims】
1. To know about the method of assay of alanine transaminase activity in serum.
2. To grasp the clinic significances of assay of alanine transaminase activity in serum.
3. To grasp the method of determination of ketone bodies in urine.
4. To understand the clinic significances of ketogenesis
5. To get more sight into the physicochemical properties of proteins.
6. To practice the assay of protein in urine.
【Periods】
3 classes
【Contents】
1. To introduce the principle of assay of ALT activity in serum , ketone bodies and protein in urine
respectively.
2. Make the standard curve
3. To assay ALT activity in serum.
4. To detect ketone bodies in urine.
5. To detect protein in urine
【Methods】
1. To assay ALT activity with standard curve.
2. To detect ketone bodies with the method of sodium nitroferricyanide.
3. To detect protein with the method of heating in acidic solution.
Experiment 11
Discussion: Substance metabolism change in serum and their clinical application
【Aims】
To give a deeper understanding of metabolism of proteins, nucleic acid, carbohydrate, lipid and vitamin,
and their clinical application.
【Periods】
3 classes
【Contents】
To discuss metabolism of proteins, nucleic acid, carbohydrate, lipid and vitamin, and their clinical
application
【Methods】
To organize the discussion.
Experiment 12
Preparation of DNA from Blood White Cells
【Aims】
To learn the technique about DNA extract from white blood cell (WBC).
【Periods】
3 classes
【Contents】
1. To introduce the principle of preparation of DNA from blood white cells
2. Isolation of WBC
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3. Breaking of WBC
4. Separation of DNA.
5. DNA storage
【Methods】
Preparation of DNA from blood white cells with phenol and chloroform.
Experiment 13
Molecular Biology VCD (Video Disc)
【Aims】
To know the common technique of molecular biology.
【Periods】
3 classes
【Contents】
1. PCR
2. Nucleic Acid Hybridization
3. Gene clone
【Methods】
To watch molecular biology VCD.
Department of Biochemistry and Molecular Biology
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