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

Pip

ette with

on

ly scale

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.)

The demands for a standard curve

Name of the curve Operator

Date

Apparatus

A

C

0

(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

Discussion

1. In which case will the protein conc. in serum decline?

2. Is it correct for your measurement of the serum protein? Why ?