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LAB 4: PROTEIN METABOLISM

I. Preparation of Standard Curve

The BCA™ Protein Assay is a detergent-compatible formulation based on bicinchoninic acid

(BCA) for the colorimetric detection and quantitation of total protein. This method combines the

well-known reduction of Cu+2 to Cu+1 by protein in an alkaline medium (the biuret reaction) with

the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+1) using a

unique reagent containing BCA. The purple-colored reaction product of this assay is formed by

the chelation of two molecules of BCA with one cuprous ion. This water-soluble complex exhibits

a strong absorbance at 562 nm that is nearly linear with increasing protein concentrations over a

broad working range (20-2,000 µg/ml). The BCA™ method is not a true end-point method; that

is, the final color continues to develop. However, following incubation, the rate of continued color

development is sufficiently slow to allow large numbers of samples to be assayed together. The

macromolecular structure of protein, the number of peptide bonds and the presence of four

particular amino acids (cysteine, cystine, tryptophan and tyrosine) are reported to be responsible

for color formation with BCA.

Because of the excellent linearity of this procedure, protein concentrations generally are

determined and reported with reference to standards of a common protein such as bovine serum

albumin (BSA). A series of dilutions of known concentration are prepared from the protein and

assayed alongside the unknown(s) before the concentration of each unknown is determined based

on the standard curve. In this exercise you will asked to determine the protein concentration of a

solution.

1. To determine the concentration of your unknown protein sample using the BCA method you

must compare the absorbance of your unknown protein to that of a known protein concentration.

Here we will utilize bovine serum albumin (BSA) to generate a standard curve. See Table 1.

2.votex for 10 seconds to allow the standard samples homogenized.

Diluted Standard Volume of protein

standard(2000μg/mL)

Volume of

Diluent (1X

PBS)

Concentration

g/mL

S1 0l 200 l 0

S2 25l 175l 250

S3 50l 150l 500

S4 100 l 100 l 1000

II. Unknown protein sample preparation 1. To ensure that the absorbance of the unknown protein sample falls within the standard

curve, you may need to prepare dilutions of your unknown.

i. You will prepare seven dilutions, see Table 2. You will determine what dilutions to make for three samples. Use Table 2 to complete your calculations.

ii. Before making the dilutions, please have an instructor check your calculations

Table 2 Dilution of unknown sample

Name Volume of

unknown sample

(µl)

Volume of

diluent (1X

PBS) (µl)

Dilution

factor

Total

volume

U1 200 0 1 200µl

U2 100 100 2 200µl

U3 50 150 4 200µl

III. Protein assay

Option 1. Semi-micro cuvette with spectrophotometer

1. ) Add 1.8 mL of working reagent (WR) to each of 200-µl dilutions (S1-S4 and U1-U3), bring the tubes up to 2.0mL.

2. ) Cover all tubes, than vortex and mix well for 10 seconds, and incubate in 37°C for approximately 30 minutes. Once time is up, be ready to read the concentration. Cool the

plate to room temperature (RT; 5 minutes on the bench top).

3. ) Turn on the spectrophotometer and set measuring wavelength to 562nm: Make sure you pick the correct cuvette (1 mL)!

4. ) Transfer 1 mL solution into the cuvette (duplicate ) 5. ) Measure and record the absorbance of the all tubes in reverse order and rinse clean the

cuvette each time after measurement (Try completing the measurements within 10-15min).

Results:

1. Absorbance reading

Tube absorbance reading at

562 nm

average absorbance

reading

S1

(Blank)

0.137

S1(Blank) 0.150 0.1435

S1

S2 0.698 0.698

S2 0.698

S2

S3 1.156 1.167

S3 1.178

S3

S4 2.016 2.046

S4 2.076

S4

U1 2.004 0.0235

U1 2.043

U1

U2 0.933 0.937

U2 0.941

U2

U3 0.715 0.7185

U3 0.722

U3

2. Construction of a Standard Curve: 1) Plot Absorbance vs concentration (µg/mL) for the BSA standards S1 through S4 to obtain

the standard curve using MS Excel. (Do not forget to standardize absorbance with S1

before making a linear regression line in excel!)

2) Use the curve fitting options to determine the linear least squares fit of the standard curve data, display the equation on your graph.

3. Unknown Protein Concentration Determination 1) Determine the trend line equation and use that to calculate the concentration for your

unknown sample.

2) ONCE YOU OBTAIN A STANDARD CURVE: OBTAIN y= mx + b formula than solve for (X). Your unknown reading goes in the Y and student should solve for X.

3) If the absorbance value of unknown samples are out of the standard value range, then they cannot be used to solve for X. Remember to multiple its diluent factor if you use a

diluted unknown sample to solve for X.

4) If there are more than one unknown sample fall in the standard value range, only one unknown sample value is needed for calculation.

4. Lab Report 1.) Follow the report format described in assignment 3.

2.) Results should include absorbance reading, standard curve, formula, and unknown

sample determination

3.) Description of graph in paragraphs

4.) Report the original unknown sample’s value.

5.) Concepts to consider for discussion:

Discuss problems that were associated with the lab (if any).

Show calculations: How do you calculate concentration of unknown from graph

and from formula given?

Interpret the meaning of value for unknown sample

y = 0.0019x + 0.046 R² = 0.9972

0

0.5

1

1.5

2

2.5

0 200 400 600 800 1000 1200

A b

so rb

a n

ce @

5 6

2 n

m

µg/ml

BSA standard curve