REPORT

Lab 5: MEASURING PROTEIN

CONCENTRATION USING
SPECTROMETER

INSTRUCTOR: Ph.D Vòng Bính Long

STUDENT NAME:
Nguyễn Trung Sơn - BEBEIU20041

Nguyễn Lê Tú Tài - BEBEIU20243

I. Introduction:

In today’s lab work, we are going to determine protein concentration by Bradford

protein assay.

Coomassie dye (Brilliant blue G250) binds to protein molecules in acid pH by two
means. The triphenylmethane group binds to nonpolar structures in proteins, and
the anion sulfonate groups interact with protein cationic side chains (e.g., arginine
and lysine side chains) in acid pH. The color change produced when the dye binds
to proteins provides a measure of total protein, which is quite sensitive in the case
of albumin and certain globular proteins. This converts Coomassie G-250 which
reaches its maximum absorbance potential at 595 nm and turns blue.

To calculate the absorbance, we use the spectrophotometer consisting of a

spectrometer to generate light of a specific wavelength and a photometer to take
the measurements of the intensity of the light. This results in the recorded
absorbance level.

II. Material and method:

-Materials and equipments includes:

MATERIALS EQUIPMENTS SAMPLE IMAGES OF

EQUIPMENTS
 Bradford Micropipettes
reagent

BSA 1 mg/mL 1 Eppendorf tubes

rack

PBS 1X/ or DW 10 Eppendorf tubes

 2 96-well plate

Microplate reader

III. Experimental procedure:

PURPOSES:

Establish a standard absorbance curve for Bovine Serum Albumin (BSA)

Experiment – Establish a standard absorbance curve for BSA This activity

involves diluting the stock BSA solution into a variety of BSA solution with lower
concentrations (ranging from 10 ug/mL to 100 ug/mL) and measuring their
absorbance level at 595 nm after exposure with Bradford reagent. The established
standard curve can be used to quantitatively determine any unknown BSA
concentration.

PROCEDURE:
Step 1. Prepare to dilute BSA in the table below:

Required volume: 1000 uL

BSA concentration (ug/mL) [0] [10] [20] [30] [40] [50] [60]
Volume of stock BSA (uL) 0 10 20 30 40 50 60
Volume of PBS 1/or DW 1000 990 980 970 960 950 940

Step 2. Add 200 uL of BSA [x] and 50 uL of Bradford reagent into each well of the
96- well plate. Mix the solution with micropipette carefully.

Step 3. Cover the plate in silver paper and incubate at room temperature for 15
minutes

Step 4. Use the microplate reader to record BSA’s absorbance (595 nm)

Step 5. From the acquired data, establish a standard curve (R2 value is
recommended to be higher than 0.95)

IV. Results and discussion:

Record of the light absorption of BSA.

[0] [10] [30] [30] [40] [50] [60] x y

0.197 0.335 0.354 0.461 0.573 0.470 0.537

0.423 0.478 0.536 0.474 0.434 0.444 0.504

0.31 0.4065 0.445 0.4675 0.5035 0.457 0.5205 0.634 0.512

0 0.0965 0.135 0.1575 0.1935 0.147 0.2105 0.324 0.202

The standard curve:

Ox: BSA concentration Oy: Optical density

We have: y = 0.004x
Unknown 1: 0.324 = 0.004[x] => [x] = 81
- BSA concentration: 81
Unknown 2 : 0.202 = 0.004[X] => [x] = 50.5
-BSA concentration: 50.5
Painting:
-y = Ax+B ● At y = 0.0965 and x = 10 ● At y = 0.2105 and x = 60
=> A = 0.0023 and B = 0.0737
-y = 0.0023x + 0.0737
+ Unknown 1: 0.324 = 0.0023x + 0.0737 => x = 108.8
-BSA concentration: 108.8
+ Unknown 2: 0.202 = 0.0023x + 0.0737 => x= 55.78
-BSA concentration: 55.78
Comparison between two results, we can see that there are errors.
Because during the experiment, we made mistakes when we added the
solution and led to the wrong result and the standard curve is not correct.
Discussion: We misused the pipette when we diluted the solution of BSA. As
a result , our result did not meet the requirements. As our group members had
taken the midterm exam then joined the lab so we were confused and hurry
that led to our carelessness and mistakes. Specifically, we overwhemingly
added BSA into the second line of the well plate before adding the solution,
and thus we also forget to add the x and y solution so we added the value of
group 3. To sum up, the higher BSA concentration that solution is, the higher
absorbance there exists.
V. Conclusion:

-After this lab experiment, we have figured out the method of Bradford assay
which measures protein concentration using a spectrometer.The color intensity of
the reaction mixture is proportional to the protein concentration in a given range.
Base on the optical absorption of the standard protein, we can determine the
protein content of the sample.The Bradford assay was successfully performed on
the microplate reader. According to the result, this protein assay is linear in the
range of 0.1 – 1.4 mg/ml. Because of its homogeneous and fast nature, the assay is
a preferred method to determine the protein concentration of samples

VI. References:
-Images of equipments:

 Adjustable volume MICROPIPETTE - 5-50ΜL. (n.d.)., from https://www.findel-

international.com/product/primary/science/glassware-plastics-and-
ceramics/micropipette-5-50l/e8r05604
 Eppendorf™ Röhrchengestell für 24 x 1.5 ml-Röhrchen For Use With Micro Test
Tubes 24 x 1.5mL to 2mL Microtube Racks | Fisher Scientific
 EPPENDORF MICROFUGE TUBE 2.0ml (KJ327-3) 500. (n.d.)., from
https://www.mcfarlanemedical.com.au/31171TP/EPPENDORF-MICROFUGE-
TUBE-2.0ML-----500/pd.php
 Bing. (n.d.)., from https://www.bing.com/images/search?
view=detailV2&ccid=tchttTXn&id=8FA77F5
0FBC76E9AC89380ABB4420B398BEC311A&thid=OIP.tchttTXnlTHKRURikV86
MgHaFV&mediaurl=https%3A%2F%2Fc.76.my%2FMalaysia%2F96-plates-flat-
bottom-sterile-10pcs-pack-goldenbiotek-1807-09- BioREV%402.jpg&cdnurl=https
%3A%2F%2Fth.bing.com%2Fth%2Fid%2FRb5c8
6db535e79531ca454462915f3a32%3Frik%3DGjHsizkLQrSrgA%26pid%3DImgR
aw&exph=649&expw=900&q=2%2B96- well
%2Bplate&simid=608038129341064920&ck=5CF78E4B30992B9010008637
2872B5CB&selectedIndex=10&FORM=IRPRST&ajaxhist=0
 PHERAstar FSX - HTS microplate reader by BMG LABTECH - THE
MICROPLATE reader COMPANY: DIRECTINDUSTRY. (n.d.)., from
https://www.directindustry.com/prod/bmg-labtech-microplate-reader-
company/product-39898-424371.html
-Bradford assay on A Microplate Reader: BMG labtech. (n.d.)., from
https://www.bmglabtech.com/bradford-assay-performed-on-bmg-labtech-microplate-
readers/