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Biochemistry Demonstration
Dept. of Biochemistry and Molecular Biology
Professor Wu Yaosheng
2012-09
Experiment One
1. Basic Demands
2. Basic Operation
3. Spectrophotometry
4. Assay of Protein Concentration
(Coomassie Brilliant Blue G-250)
1. Basic Demands
To abide the rules in laboratory◆Attendance at practicals is compulsory◆Always wear a laboratory coat during experiment process◆Do not smoke, eat, chew, drink in the lab◆Keep your area of bench tidy and organized◆When handling hazardous solution or gas, using ventilati
on cabinet ◆ Experiment reports are required and submitted by the du
e date◆Keep the rules for the students on duty at the end of exp.
Experiment report demands
Name of the experiment
Date of the experiment
Principle
Protocol
Results (phenomenon and data )
Calculate
Analysis and discussion
2. Basic Operation
Pipeting
Automatic pipettes
Stir and blend
Clean the glass vessel and graduated pipettes after used
3. Spectrophotometry
Principle:
Beer-Lambert law
A=kCL
Absorbance
Constant
concentration
path
len
gth o
f light
Absorbance ( optical density or extinction)
Absorbance is measured by the spectrophotometer
A = log10
I0
I
I0 ----the intensity of the incident light
I0 I
I ----the intensity of the transmitted light through the solution
3. Spectrophotometry
Principle:
Beer-Lambert law
A=kCLA is proportional to C of the solution
5% CuSO4 solution, copper sulfate, bluestone
(1) To calculate the Conc. of an unknown solution using standard tube
Standard solution : As=KsCsLs (1)
Unknown solution : Au=KuCuLu (2)
(1)/(2), AsAu
= CsCu
Cu = AuAs
× Cs
(2)To obtain the Conc. of unknown solution by standard curve method
Tube Blank 1 2 3 4 5
Conc. C1 C2 C3 C4 C5 C6
Absorbance
A1 A2 A3 A4 A5 A6
A
C0
X-coordinate Y
-coordinate
The demands for a standard curve
Arrangement in reason
Indicate unit for concentration
Other information( the name of the curve, the operator, the date, apparatus or machine used, et al.)
(2)To obtain the Conc. of unknown solution by standard curve method
Tube Blank 1 2 3 4 5 unkown
Conc. C1 C2 C3 C4 C5 C6 Cu?
Absorbance
A1 A2 A3 A4 A5 A6 Au
A
C0
Au
Cu
4. Assay of Protein Concentration (Coomassie Brilliant Blue G-250)The methods for the measurement of protein c
ontent in samples
To determine the nitrogen content
Spectrophotometry
Ultraviolet spectrophotometry
Visible light spectrophotometry
( various Colorimetric reaction )
Coomassie Brilliant Blue G-250
Aims:
To learn how to assay protein conc. Using Coomassie Brillilant Blue G-250 dyeing method
Principle:
Coomassie Brilliant G-250 + Pr
Colour of the solution changed from yellow to blue
H+
Absorbance peak is at 595 nm
Protocol
1. Make a standard curve
Reagent 1 2 3 4 5 6 Unkown
Pr standard
(0.1mg/ml)
0.1 0.3 0.5 0.7 0.9 0 Sample 0.1
NaCl (9g/L) 0.9 0.7 0.5 0.3 0.1 1.0 0.9
Pr reaction solution
5.0 5.0 5.0 5.0 5.0 5.0 5.0
Coresponding Pr conc. (mg/ml)
0.01 0.03 0.05 0.07 0.09 0 ?
A595 0
( mlL )
Coomassie Brilliant Blue G-250 standard curve for protein assay
Operator
Date
Apparatus
0
A595
C mg/mL
C g/L0.02 0.04 0.06 0.08 0.10
0.1
0.2
0.3
0.4
0.5
Protocol 2. Assay of the blood serum specimen
(1) Dilute 200 times for the serum (has been done by technician in lab.
(2) Manipulate the diluted sample following the method of making standard curve.
(3) Find the protein concentration from the standard curve according to A595.
Calculation Blood serum protein conc. (g/L)
= data located from the standard curve × 2000.1
200----diluted times
0.1----the volume of dilution serum sample
Normally, reference value extent of serum protein for an adult:
60~80g/L