Scientific and calculate the concentration of protein by using

Scientific Communication and Laboratory Skills

 

Lab
Report

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Abstract

 

Bradford
protein assay and direct ultraviolet absorption (UV light) were performed on
unknown samples. These two methods were performed to determinate the
concentration of the unknown samples.

 

The Bradford
assay is colorimetric assay depends on the reaction between Coomassie brilliant
blue and the aromatic residues in the protein. After the dye react with these
residues it increases the absorbance from 470nm to 595nm. In general, absorbance
of a serious of known concentrations of standard protein were measured and
create a standard curve. use the created standard curve to calculate the
concentration of unknown sample based on its absorbance. The method is highly
sensitive, and it is very fast but small materials can interfere with it and
highly effect the results.

 

The UV absorption
can be applied on protein that contains aromatic residues such as tyrosine,
tryptophan. There aromatic residues are able to absorb the UV light at 280 nm
without adding colouring reagent. uv spectrometer is used to measure the
absorbance and calculate the concentration of protein by using beer’s law. Extinction
coefficient for the protein is required to carry on the calculation. The
technique is quick but the successful of the method depends on having the
accurate extinction coefficient for the protein.

The aim of
using the both methods is to compare between its results and show if there any
different between them.

 

introduction

 

protein assays
are one of the most widely used methods in life science research. Knowing
protein concentration is required in protein purification, cell biology,
electrophoresis and other research applications, it is important to use more
than once method during experiment to obtain the require accurate results.
there are wide variety of methods such as dye binding assays (Bradford), copper
ion based assay (lowry and BCA), and spectrophotometry based on UV absorption.

Each protein
assay has it is advantage and limitations and it shows the reliability of the
method. The natural of the sample is the most important consideration to choose
the right assay method. If the sample is in solid form it can be easily
solubilized in protein assay but most of the protein samples are complex
solutions which include non-protein materials. There is many issues effect the
reliability of the methods such as interfering agents, sample preparation,
assay sensitivity, sample size and time consideration.

 

Proteins are
complex polymers of amino acids with huge range of modification and different structures
which required numerous numbers of chemical agents to stabilise it. Non-protein
agents can be a challenge for protein assays, protein solution contains
surfactant can interfere with dye based assay as Bradford assay. The better
reliability will be possible with removing non-protein assays or by using
method which overcome the interfering effect of it.

The size of
the sample becomes a critical consideration, as most of the methods required at
least 0.5 µg of proteins for a reliable result, the method that requires
the smallest amount of protein will offer an advantage over the others such as
dotMETRIC assay. The amount of the time which taken to complete the experiment
will depend on the complexity of the sample and the assay method. The methods
which use standard plots and curves are faster while the protein sample
contains non-protein agents are consuming more time which needed to remove the
non-protein agents.  The assays that does
not required standard plots can be faster and cheaper such as protein dotMETRIC
protein assay and dye binding CB-X.

 

The most reliable protein estimation is the one which
performed using protein standard. The protein standard should have similar
properties to the sample that being tested. It is very difficult to find
protein standard with similar properties to the sample. Bovine serum albumin
and gamma globulin are accepted to use as reference protein.

As in this
experiment, bovine serum albumin was used to create a serious of standard
solutions with known concentrations. Two spectroscopic methods were using to
obtain the absorbance of the standard solutions. The absorbance and the
concentration of the standard solution were used to create calibration curve to
detect the concentration of the unknown samples. The result of the two
spectroscopic methods will be compared to discover if there any difference
between them.

 

Materials and methods

 

Bradford essay

 

By using 20?l
from each different solution mix with 1ml of the Coomassie reagent in an Eppendorf
tube, mix it on a vortex and leave it at room temperature for 5 minutes.

A blue colour should start to develop once the protein
solution will be mixed with the reagent. The power of the colour will be
related to the protein concentration in the sample.

The unknown samples will be treated in the same way the
other sample reagent with the same method. In addition of a blank has prepared
to calibrate the spectrophotometer at the wavenumber 595nm.

Record all the results in a table.

 

 

Ultraviolet light

 

In this experiment will not be added any reagent to generate
the colour needed because the protein can absorb the ultraviolet radiation at certain
wavelength and that is because of the aromatic amino acid, such as
phenylalanine, tyrosine and tryptophan.

Set the spectrophotometer will be set at 280nm, it will be
calibrated with distilled water. Cuvettes that are compatible with UV light
were used in this method.

After the calibration, the solution will be measured and recorded.
that experiment must be repeated 3 times and the results can be presented in a
table.

Materials used in the
experiments

Protein (Bovine serum albumin) stock solution 1 mg/ml

Unknown solution W, X, Y, and Z

Coomassie (Bradford) Protein Assay Reagent (Thermo
Scientific)

Distilled water

Eppendorf microtubes

Visible light cuvettes

Phosphoric acid

 

 

Safety assessment and precaution: this experiment was conducted in level two
laboratory. Bradford assay contain methanol and low concentration phosphoric
acid which handle in container in hood with away from eye, skin or any material
can be flammable.

As the chemical can be
irritating or toxic it has to be handle in a specific way gloves must be wear
all the time so as safety glasses and lab coat.

 

 

Results and discussion

 

preparation of
protein standard solutions.

Bovine serum albumin in water 0.5%

It means 0.5g in each 100 ml = 0.005g/ml

5000 ?g/ml

Total volume =
protein stock solution + distilled water = 2000 µl

Convert µl to ml dividing by 1000

2000 µl/ 1000
= 2ml

Use the following equation to find the concentration of the
standard solution

C1 V1 = C2 V2

C2 = C1 V1 / V2

 

Stander solution 1

Protein stock solution 50 µl = 0.05 ml

C2 = 5000x 0.05 / 2 = 125 ?g/ml

 

Stander solution 2

Protein stock solution 100 µl = 0.1 ml

 C2 = 5000x
0.1 / 2 = 250 ?g/ml

 

Stander solution 3

Protein stock solution 200 µl = 0.2 ml

C2 = 5000x 0.2 / 2 = 500 ?g/ml

 

Stander solution 4

Protein stock solution 300 µl = 0.3 ml

C2 = 5000x 0.3 / 2 = 750 ?g/ml

 

 

 

Stander solution 5

Protein stock solution 400 µl = 0.4 ml

C2 = 5000x 0.4 / 2 = 1000 ?g/ml

 

 

 

Standard

 

1

2

3

4

5

 

 

 

solution

 

 

 

 

 

 

 

 

 

 

 

 

 

Protein
stock

 

50

100

200

300

400

 

 

 

solution (?l)

 

 

 

 

 

 

 

 

 

 

 

 

 

Distilled water

 

1950

1900

1800

1700

1600

 

 

 

?l

 

 

 

 

 

 

 

 

 

 

 

 

 

Concentration

 

 

 

 

 

 

 

 

 

?g/ml

 

125

250

500

750

1000

 

 

 

 

 

Bradford
assay

 

 

This table shows
the absorbance values for standard and unknown solutions in Bradford assay

 

 

solution

1

2

3

4

5

X

Y

Z

 

 

Absorbance

 

0.069

0.092

0.232

0.320

0.410

0.215

0.267

0.352

 

 

 

0.065

0.095

0.243

.0326

0.405

0.214

0.271

0.349

 

 

at 595 nm

 

 

 

 

0.072

0.099

0.235

0.331

0.411

0.218

0.275

0.359

 

 

 

 

 

 

 Calculate the average absorbance and standard
deviation for each standard solution and unknown samples using data given in
table

 

 

Solution 1

Average = (0.069 + 0.065 + 0.072) / 3 = 0.069

Calculation of Stander deviation:

Calculate the mean (0.069).

For each number: subtract the mean. Square the result.

Add up all the squared results.

Divide this sum by one less than the number of data points (N –
1). This gives you the sample variance.

Take the square root of this value to obtain the sample standard
deviation.

(0.069 – 0.069)2 = 0

(0.065 – 0.069)2 = 0.00016

(0.072 – 0.069)2 = 0.000009

(0 + 0.00016 + 0.000009) /2 = 0.0000125

Standard deviation = ? 0.0000125 =0.0035

Solution 2

Average = (0.092 + 0.095 +0.099) / 3 = 0.096

Standard deviation =0.0035

Solution 3

Average =(0.232 + 0.243 + o.235) / 3 = 0.236

Standard deviation= 0.0051

Solution 4

Average = (0.320 + 0.326 + 0.331) / 3 = 0.325

Standard deviation = 0.0055

Solution 5

Average = (0.410 + 0.405 + 0.411) / 3 = 0.409

Standard deviation = 0.0032

Solution X

Average = (0.214 + 0.215 + 0.218) / 3 = 0.216

Standard deviation = 0.0021

Solution Y

Average = (0.267 + 0.271 + 0.275) / 3 = 0.271

Standard deviation = 0.004

Solution Z

Average =(0.352 + 0.349 + 0.359) / 3 = 0.353

Standard deviation = 0.005

table that
shows the concentration of each standard solution and its corresponding average
absorbance.

 

Standard

1

2

3

4

5

Concentration

125

250

500

750

1000

?g/ml

 

 

 

 

 

Average

0.069

0.096

0.236

0.325

0.409

Absorbance
at 595 nm

 

 

 

 

 

calibration
curve of absorbance against concentration of the standard solutions.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Using the
calibration curve and its regression equation, calculate the concentration of
the unknown samples. Use the average absorbance for each unknown in your
calculation and report your answers in ?g/ml (to 2 decimal places) as well as
in w/v% and ppm.

 

From the
calibration curve  

y = mc + b

Y = 0.0004X +
0.0144

X = (Y –
0.0144) / 0.0004

 

Solution X

0.216 =
0.0004X + 0.0144

X = (0.216 –
0.0144) / 0.0004 = 5.04 x102 ?g/ml (to 2 decimal places)

w/v%

first convert
?g to gram then multiple by 100

5.04 x102
?g / 106 = 0.000504 g/ml

w/v% = 0.0504%

ppm= 504 ppm

 

 

 

 

 

Solution Y

X = (0.271 –
0.0144) / 0.0004 = 64.15 x10 ?g/ml (to 2 decimal places)

w/v% = 0.06415%

ppm = 641.5
ppm

 

 

solution Z

X = (0.353 –
0.0144) / 0.0004 = 86.45 x10 ?g/ml (to 2 decimal places)

w/v% 0.08645%

ppm = 864.5
ppm

 

standard

X

Y

Z

Concentration
?g/ml

504

641.5

864.5

Average
absorbance at 595 nm

0.216

0.271

0.353

 

 

 

 

 

 

 

 

Ultraviolet
light

 

Table shows
absorbance values for standard and unknown solutions at 280 nm

 

 

solution

1

2

3

4

5

X

Y

Z

 

 

Absorbance

 

0.112

0.205

0.415

0.612

0.795

0.421

0.525

0.541

 

 

 

0.105

0.211

0.427

0.628

0.812

0.409

0.532

0.522

 

 

at 280 nm

 

 

 

 

0.111

0.217

0.422

0.625

0.829

0.414

0.515

0.536

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

the average
absorbance and standard deviation for each standard solution and unknown
samples using data given in table

 

 

 

Solution 1

Average = (0.112 + 0.105 + 0.111) / 3 = 0.109

Standard deviation = 0.0038

 

Solution 2

Average = (0.205 + 0.211 +0.217) / 3 = 0.211

Standard deviation =0.006

 

Solution 3

Average =(0.415 + 0.427 + 0.422) / 3 = 0.421

Standard deviation= 0.006

 

Solution 4

Average = (0.612 + 0.628 + 0.625) / 3 = 0.621

Standard deviation = 0.0085

 

Solution 5

Average = (0.795 + 0.812 + 0.829) / 3 = 0.812

Standard deviation = 0.017

 

Solution X

Average = (0.421 + 0.409 + 0.414) / 3 = 0.415

Standard deviation = 0.006

 

 

Solution Y

Average = (0.525 + 0.532 + 0.515) / 3 = 0.524

Standard deviation = 0.0085

 

Solution Z

Average = (0.541 + 0.522 + 0.536) = 0.533

Standard deviation = 0.0098

 

 

table that
shows the concentration of each standard solution and its corresponding average
absorbance. For example:

 

Standard

1

2

3

4

5

Concentration

125

250

500

750

1000

?g/ml

 

 

 

 

 

Average

0.109

0.211

0.421

0.621

0.812

Absorbance
at 280 nm

 

 

 

 

 

 

 

calibration
curve of absorbance against concentration of the standard solutions.

 

 

 

Using
the calibration curve and its regression equation, calculate the concentration
of the unknown samples. Use the average absorbance for each unknown in your
calculation and report your answers in ?g/ml (to 2 decimal places) as well as
in w/v% and ppm.

 

From the
calibration curve 

y = mc + b

Y = 0.0008X +
0.0144

X = (Y –
0.0144) / 0.0008

 

Solution X

0.415 =
0.0008X + 0.0144

X = (0.415 –
0.0144) / 0.0008 = 500.75 ?g/ml (to 2 decimal places)

w/v%

first convert
?g to gram then multiple by 100

500.75 ?g / 106
=  0.00050075 g/ml

w/v% = 0.050075%

ppm= 500.75
ppm

 

Solution Y

X = (0.524 –
0.0144) / 0.0008 = 6.37 x102 ?g/ml (to 2 decimal places)

w/v% = 0.0637%

ppm = 637 ppm

 

 

solution Z

X = (0.533 –
0.0144) / 0.0008 = 648.25 ?g/ml (to 2 decimal places)

w/v% = 0.08645%

ppm = 652 ppm

 

standard

X

Y

Z

Concentration
?g/ml

500.75

637

648.25

Average
absorbance at 280 nm

0.415

0.524

0.533

 

 

Conclusion

 

Through
the experiment, we will able to find out the concentration of the unknown samples
by using calibration curve of absorbance against concentration. From the
standard curve of Bradford assay, the unknown solutions (X,Y,Z) have
concentration of 504 µg/ml , 641.5 µg/ml and 864.5µg/ml. while the
standard curve of UV light absorption shows a similar concentration for sample
(X,Y) 500.75 µg/ml and 637 µg/ml but different concentration for sample
(Z) 648.25 µg/ml and that can be because of interfering agents, sample preparation, and assay sensitivity.

 

Both Bradford
assay and UV light absorption show that the absorbance has direct relationship
with the concentration.

 

 

 

 

 

 

 

 

 

 

 

References

 

·               
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities
of protein utilizing the principle of protein-dye binding. Anal Biochem 72:
248-254.

·               
Kalb, V. F. and Bernlohr, R. W. (1977). A new
spectrophotometric assay for protein in cell extracts. Analyt. Biochem. 82, 362–371.

·               
Kirschenbaum, D. M. (1975) Molar absorptivity and A1%/1 cm
values for proteins at selected wavelengths of the ultraviolet and visible
regions. Analyt. Biochem. 68, 465–84.

·               
Stoscheck, C. M. (1990). Quantitation of protein. Methods Enzymol 182: 50-68.

·               
Spector, T. (1978) Refinement of the Coomassie Blue method
of protein quantitation. A simple and linear spectrophotometric assay for