Biochemistry and molecular biology lab MANIK

Biochemistry and Molecular Biology Lab
Prepared By- Md. Imran Nur Manik
BIOCHEMISTRY AND MOLECULAR BIOLOGY LAB
Table of Contents
Experiment Number -01.........................................................................................1
Name of the experiment: Estimation/quantization of protein by
spectrophotometric method...............................................................................1
Experiment Number -02.........................................................................................5
Name of the experiment: Determination of extinction co-efficient of BSA
(Bovine serum albumin, 67000 Dalton) ...........................................................5
Experiment Nubmer-03..........................................................................................8
Name Of The Experiment: Determination of serum triglyceride ...............8
Experiment Number: 04.......................................................................................10
Name of The Experiment: Determination of amount of urea in blood
serum ...................................................................................................................10
Md.
Imran
Nur
Manik

Arranged By- Md. Imran Nur Manik, B.Pharm.;M.Pharm.; RU Page 1
Prepared by-Shadid UZ Zaman At Tadir; B.Pharm.; M.Pharm.; DU
Experiment Number -01
Name of the experiment: Estimation/quantization of protein by
spectrophotometric method.
1.1. Principle:
Proteins are polymer of amino acids which have definite shape and absorb light of definite
amount. Spectrophotometer is an instrument which can generate light of different wavelength,
have space to lap substance of whose absorbance is to be determined and screen to visualize the
absorbance. If light of definite wavelength is passed through the BOVAIN SERUM ALBUMIN
(BSA), a definite amount of wavelength is absorbed and remaining amount is transmitted which
is called transmittance. For a particular sample there is a wavelength in which there will be
maximum absorbance. This wavelength is called λmax. It is determined by placing the sample in
cubet & spectrophotometer is set to generate & pass light of different wavelengths. In the screen
there will be formation of a graph from which the maximum wavelength, at which there is the
maximum absorbance, is determined. The wavelength is used in experimental purposes. Protein
present in BSA is expressed in mg/mL unit. Absorbance (A) by the sample at the λmax
wavelength is expressed as:-
cA
abcA

,
alter)may(whichionConcentratc
Constant)cm,1(usuallylengthPathb
ility)(absorptibConstanta
AbsorbanceA
Here,




i.e., absorbance is directly proportional to the concentration of BSA in the sample.
A point to be noted that absorbance is due to the presence of Tyrosine & Tryptophan, which
mainly absorb light of maximum wavelength. So to stabilize and further handling of the bovine
serum solution, it is essential to keep them in buffer solution which is prepared by KH2PO4 and
K2HPO4 in equimolar amount.
This buffer has enough capacity to keep the solution stable. BSA of different
concentration is placed in cubet from which different absorbance is obtained. This absorbance is
plotted against concentration of the solution which is later imputed in computer Microsoft
Office Spreadsheet program, MS Excel in order to obtain absorbance curve. This will yield the
equation of the straight line and value of R2
, the nearer the value of R2
to 1 the better the
experiment. Supplied samples taken and the absorbance of that are determined to place the
value in the absorbance equation to determine the concentration of the sample.
The equation may be:-
m
c
Yx
cmxY


i.e.,
Md.
Imran
Nur
Manik

1.2. Apparatus:
1. Spectrophotometer
2. Computer having MS Excel
program
3. Test tube and test4ube rack
4. Micropipette
1.3. Reagent:
1. BSA(Bovine Serum Albumin)
2. KH2PO4
3. K2HPO4
1.4. Procedure:
1.4.01. Preparation of buffer:
Phosphate buffer of pH- 7.4 was prepared by taking equimolar amount of the K2HPO4
and KH2PO4. The molecular weight of KH2PO4 and K2HPO4 are 136 and 174 respectively. 50
m.mol of each of the substance was taken in order that
KH2PO4 requires, 13650/1000=O.68gm =680mg
K2HPO4 requires, 17450/1000 =0.87gm =870mg
680mg of KH2PO4 and 870mg K2HPO4 was taken simultaneously and placed initially in little
amount containing volumetric flask. By shaking the substances they were dissolved and made
the volume up to 100 ml.
This is the buffer of pH-7.4, which was used with the unknown concentration sample and
concept handling known concentration of BSA.
1.4.02. Preparation of BSA solution of different concentration:
In order to prepare graph of absorbance against concentration, varying amount of BSA
solution (supplied from lab holding concentration 1mg/mL) and phosphate buffer were
prepared, to have preparation containing 0, 125, 250, 500, 750, l000mg/mL BSA.
The different amount of different substances was taken was taken as expressed in the
following table.
Estimation/quantization of protein by spectrophotometric method
Sample No. BSA (mL) Buffer (mL) Concentration of BSA (µg/mL)
01
02
03
04
05
06
07
Varying amount of different noted substance were taken in 6 test tubes and kept standing
for some time.
Md.
Imran
Nur
Manik

1.4.03. Determinations of λmax and absorbance of the sample:
a) By supplying electricity or other equipment the Spectrophotometer was turn on.
b) The cubet was washed with the buffer and the outside was cleaned by soft tissue paper.
c) 1mL of the buffer solution was taken in cubet and program set to determine
the λmax from 180-500 nm.
d) From the plot visualization in the screen of the spectrophotometer the λmax was 280 nm
i.e. the maximum absorbance would give is 280nm and the absorbance of the sample
2,3,4,5 and 6 were taken.
e) This absorbance corresponded to the concentration of serum in the sample.
The absorbance of the sample is in the following table:
Sample Concentration of BSA (µg/mL) Absorbance
01
02
03
04
05
06
07
Table 01.02: Absorbance at different concentration
Using computer Microsoft office excel, plot of absorbance against concentration of the
corresponding solution yielding the following equation:
Y
i.e. concentration of the supplied sample = (Y-)
And, 2
R
i.e. the equation justifies its stand with enough strength.
1.5. Result:
Only the procedure was studied, no sample was given. If it was given by determining the
absorbance concentration of the sample would be determined by putting this absorbance in
equation.
1.6. Precaution:
1. The test tube, micropipette, measuring flask should be clearly washed before use.
2. The spectrophotometer weight should be standardized prior to use.
3. Before taking prior out of the graph, the zoom of the page should be observed
carefully.
Md.
Imran
Nur
Manik

y = 0.0005x + 0.0195
R² = 0.9858
0
0.1
0.2
0.3
0.4
0.5
0.6
0 500 1000 1500
Absorbance
Conc.
Determination of protein content by
UV-spectrophotometric method
Series1
Linear (Series1)
Md.
Imran
Nur
Manik

Experiment Number -02
Name of the experiment: Determination of extinction co-efficient of BSA
(Bovine serum albumin, 67000 Dalton)
2.1. Principle:
Extinction coefficient is constant in the Beer-Lambert law of absorption equation when
concentration term is expressed in terms of mole/litre. In this case coefficient is known as molar
extinction coefficient and expressed by ε, the Beer-Lambert law is-
A = abc………. (1)
Here,
A = Absorbance
a = absorptivity constants
b = path length (1cm, constant)
c = concentration
If the concentration term is expressed by mol/ litre, (1) yields-
)2........(
bc
A
bcA




ε = Extinction coefficient or molar extinction coefficient
Supplied BSA was taken and different amount (µg/mL) was taken in test tube & equal mole of
KH2PO4 & K2HPO4 was taken to prepare buffer to stabilize the BSA and different amount of
this buffer preparation was added to the test tubes to impart same quantity in terms of volume
but different concentration of terms of weight of the BSA.
The sample was handled towards spectrophotometer to bring out the absorbance in different
concentrations which were used to plot a graph of absorbance against concentration in order to
prepare a straight line and its equation.
The value of the R2
of this equation expresses the strength of the equation.
The absorbance of known concentration was determined, concentration was expressed in terms
of mol/litre and putting these values in equation (2) yielded the extinction co efficient of the
equation. This extinction co efficient expresses the absorbance of 1 molar solution of BSA.
2.2. Apparatus:
2. Computer having MS program
3. Tart Tubes of Test tube rack
4. Micropipettes and tips
2.3. Reagent:
1. BSA
2. KH2PO4
Md.
Imran
Nur
Manik

2.4. Procedure:
2.4.01. Preparation of buffer:
Prospect buffer 7.4 was prepared lay taking equimolar amount of both of the K2HPO4 and
KH2PO4 having the molecular weights of K2HPO4 and KH2PO4 are 136 and 174 respectively.
50 mol of each of the substrate was taken for that
KH2PO4 requires, 136X50/1000 = 0.68gm = 680mg
K2HPO4 requires, 174X50/1000 = 0.87gm = 870mg
And 680 mg of KH2PO4 & 870 mg of K2HPO4 were taken simultaneously and lodged in
initially little amount containing volumetric flask by shaking the substances were dissolved and
make the volume up to 100 ml this is the buffer of pH 7.4 which was used with the sample of
different concentration.
2.4.02 Preparation of BSA solution of different concentration:
In order to prepare Graph of Absorbance against concentration varying amount of BSA
solution (prepared from sample of concentration 1 mg/mL supplied from laboratory) and
phosphate buffers were prepared to have preparation containing 0, 125, 250, 500, 750, 1000
mg/mL BSA.
The different amounts of different substances were taken as expressed in the following table:
SAMPLE BSA (mL) BUFFER (mL) CONCENTRATION OF BSA (µg/mL)
01
02
03
04
05
06
07
Table-2.1: Different samples containing varying of BSA
The substances were taken in 6 test tubes and were kept standing in the test-tube rack.
2.4.03. Determination of λmax:
a) The spectrophotometer was turned on.
b) The cubet was washed with buffer and the outside was cleaned by soft tissue paper.
c) Buffer was taken in the cubet and the spectrophotometer was adjusted to 0.
d) The sample 2 was taken and set the program to determine the λmax at 180-500nm.
e) From the graph visualized in the screen of the spectrophotometer the λmax was
determined 280nm, i.e., 280nm is the wavelength at which maximum absorbance of the
sample (BSA) would be given.
Md.
Imran
Nur
Manik

2.4.04. Determination of absorbance, straight-line and value of R2
:
The spectrophotometer was adjusted to 280nm and absorbance of sample 2,3,4,5, & 6 were
determined. This absorbance corresponded to the concentration of serum in the sample. The
following table contains the absorbance of different concentration.
Sample Concentration (µg/mL) absorbance
1
2
3
4
5
6
7
Table-2.2: Absorbance at different concentration
Using Microsoft office excel, plot of absorbance against concentration of the corresponding
solution yield the following equation


2
R
Y
2.5. Calculation:
Absorbance, A =
Concentration, c =
Path length, b =
Now,
From the equation (2), we get,
Extinction coefficient,
bc
A

=
=
2.6. Result:
The extinction coefficient of BSA is= M-1
cm-1
2.7. Precaution:
1. The syringe should be sterile and it is best to use new syringe.
Md.
Imran
Nur
Manik

Experiment Nubmer-03
Name Of The Experiment: Determination of serum triglyceride
3.1. Principle:
Tri glycerol is any combination of Glycerol with three different fatty acids. These substances,
triacyglycerols, are also called neutral fats. In the blood triacyglycerols combine with proteins to
form lipoprotein. The liver synthesizes lipoprotein to transport fats to other tissues where they
are a source of energy. Fat in adipose is stored energy. Triglyceride in blood can be determined
by using the principal of Beer-Lambart’s law of absorbance in spectrophotometer. The reaction
takes place between the reagent and the HDL so the absorbance varies due to variation of
amount of TAG. By comparing this absorbance with a standard the amount of TAG is
determined.
3.2. Reagent:
a) Triagiycerol kit
b) Standard for determination of triglyceride.
c) Blood serum
3.3. Apparatus:
a) Spectrophotometer
b) Centrifuge machine
c) 3 test tubes and test tube rack
d) Sterile syringe.
3.4. Procedure:
3.4.01. Preparation of serum:
With the help of laboratory assistant blood was taken by syringe from a healthy person to
centrifuge tube and later was placed in the centrifuge machine for centrifugation.
3.4.02. Preparation of sample:
Three test tubes were dried after washing and placed in the test tube rack. They were labeled as
BLANK, SAMPLE and STANDARD. Different amount of different substance were taken in
the test tubes as described in the table.
The test tubes were kept standard for 10 minutes.
REAGENT BLANK (µL) STANDARD (µL) SAMPLE (µL)
Reagent kit
Standard
Serum
Table3.1: Different substance in different amount in 3 test tubes
Md.
Imran
Nur
Manik

3.4.03. λmax Determination:
It was information provided from the laboratory that λmax for determining TRIGLYCERIDE is
530. If it was not informed λmax could be determined by setting of spectrophotometer to
determine absorbance from 200-600 nm. The pick point of the graph was 530nm. So the λmax is
530 nm.
3.4.04. Determination of absorbance the sample and standard:
The cubet was filled with the reagents taken from test tube 1. Then the spectrophotometer was
adjusted to zero. The cubet was again washed and filled with the sample and absorbance was
determined. Lately the cubet was washed and finally filled with standard. The absorbance was
taken.
3.5. Calculation:
The absorbance (optical density) of the sample is=
The absorbance (optical density) of the standard is=
So the concentration of triglycerides = standardofionconcentrat
standardofOD
sampleofOD

=
=
3.6. Result:
The concentration of tri glycerol in blood is . It is value for an
adult person.
3.7. Precautions:
1. The test tubes, micropipette, tips, measuring flask should be clearly washed.
2. The spectrophotometer, weigh should be standardized prior to use.
3. Before taking print out of the graph, the scale of the page and other equipment should
be adjusted properly.
Md.
Imran
Nur
Manik

Experiment Number: 04
Name of The Experiment: Determination of amount of urea in blood serum
4.1. Principle:
Urea is diamide of carbonic acid, a crystalline solid having formula CH4N2O, found in
blood, lymph and urine. It is formed in liver from ammonia derived from the deamination of
amino acids. It is the chief nitrogenous constituent of urea and along with carbon dioxide, the
final product of protein metabolism in the body. In normal condition , urea represents 80-90%
of the total urinary nitrogen The normal value of urea in blood is 15 to less than 50 uL/dL.
Increased amount of urea indicates the higher intake of protein in the diet .lf still higher amount
of urea it indicates the failure of kidney or dysfunctional kidney. The increased accumulation of
urea in blood is called ureamia, one of the consequences of kidney disorder. Determination of
urea in blood thereby can also be applied to see where there is any disorder in kidney or not.
Spectrophotometer is used to determine the optical density and λmax for the sample.
Dilution factor is multiplied the ratio of optical density (OD) of sample and standard.
Serum urea concentration= standardofionconcentrat
standardofOd
sampleofOD

4.2. Apparatus:
2. Test tubes & test tube rack
3. Sterile Syringe
4. Centrifuge machine
4.3. Reagent:
1. Reagent I
2. Reagent II
3. Blood Serum
4.4. Procedure:
4.4.01. Collection of plasma:
Blood taken with the help of laboratory assistant by a sterile syringe (it was recommended &
used a new syringe). It was attributed for 10 minutes at 3000 rpm. Plasma was repeated from
the blood cells and was supernatant solution. It was the plasma on which reagent, enzyme
would be added.
4.4.02. Preparation of the sample:
3 test tubes were taken and labeled as blank, standard and sample. Then following
amount of different substances were added as the table bellow
Blank Standard Sample
Reagent 1
Enzyme
Distilled water
Standard
Reagent 2
Sample
Md.
Imran
Nur
Manik

4.4.03: Different amount for determining urea.
The test tubes were kept for some time about 10 min and µL reagent II was added to each
of them.
4.4.04: λmax determination:
Spectrophotometer was turn on and program was to determine from 400-700nm wavelength. In
the cubet sample blank was taken and adjusted to zero and then sample was taken to determine.
It found to be 570nm.
4.4.05: Optical density of the sample and standard
Since by placing blank in cubate machine was adjusted only sample was again placed against
570nm wavelength to determine the absorbance of the sample. Finally cubet was washed with
the standard or sample which was supposed to taken reading and was cleared by using soft
tissue.
4.5. Calculation:
Optical density of the sample =
Optical density of the standard =
Serum urea = standardofionConcentrat
sampleofOD
sampleofOD

=
=
5.5. Result:
The serum urea of blood urea is L4tE5rpg/I
5.6. Precaution:
1. The syringe must be sterile
2. The reagent is small in amount so micropipette should be used
3. Before taking the sample. Standard or reagent by micropipette every time a new
tip should be used to avoid unwanted mixture
Md.
Imran
Nur
Manik

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