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Calculation of Unknown Protein Sample Concentration

Tube

S1 (Blank)

0.137

0.1435

S1(Blank)

0.150

S2

0.698

0.698

S2

0.698

S3

1.156

1.167

S3

1.178

S4

2.016

2.046

S4

2.076

U1

2.004

2.0235

U1

2.043

U2

0.933

0.937

U2

0.941

U3

0.715

0.7185

U3

0.722

The standard curve was made by S1,S2,S3,S4(which have both concentration and absorbance). We add a trendline for this scatter plot, add the equation and R^2 for the trendline. R^2 is used to test if the model is fit or not. The closer to 1, the fitter model we get.

Now, we need to calculate the unknown samples (U1,U2,U3). We have U1, U2,U3’s absorbance. Now we need to calculate there concentration. The trendline will be used here.

Y=0.0019x+0.046
For U1, 2.0235=0.0019x+0.046
X=1040.789 ug/ml

For U2,
0.937=0.0019x+0.046
X=468.9474ug/ml

For U3,
0.7185=0.0019x+0.046
X= 353.9474ug/ml

Now we get the all the concentration of U1,U2 and U3.

How to make standard curve?

1 Input your data in excel

2 select all the data and insert the Scatter charts

3 Double-click one of the points in the chart, it will show some options

4 Click Add Chart Element-Trendline- Linear

5 Click Display Equation on chart, and display R-squared value on chart

6 In this graph, x-axis is concentration, y- axis is absorbance. Use the equation to calculate your unknow samples’ concentration, which is you know y and solve x.

LAB 4: PROTEIN METABOLISM

PURPOSE:

To employ proper laboratory techniques in order to determine whether one is in positive,

negative, or zero nitrogen balance by knowing the protein (nitrogen) intake and the

urinary urea nitrogen levels of a 24 hour urine collection.

BACKGROUND:

Proteins are complex organic compounds that are necessary for growth and repair of

all living matter. They are formed from smaller structural units called amino acids. Amino

acids are bonded together to form a “string” which then folds and coils upon itself giving

the protein its characteristic shape and function. There are 20 different amino acids used

in proteins, 9 of which are considered nutritionally essential. Amino acids are all alike in

the respect that each one contains a central carbon atom (C), an amine group (NH2), a

carboxyl group (COOH), and a radical group (R). Proteins differ because the radical group

is different for each amino acid.

H

|

NH2– C – COOH

|

R

When we ingest protein, our body dismantles the dietary protein into its amino acid

components that can be absorbed in the small intestine. Inside the body’s cells, the amino

acids are reassembled into proteins specific to our body such as: enzymes, hormones,

structural material, etc.

Although it is considered “biologically expensive”, proteins can also be used to provide

energy. When excess proteins or amino acids are ingested, the amino acids that are not

used as building blocks are deaminated. Once the amine group is removed, it is excreted

through the urine as urinary urea nitrogen (UUN). The remaining molecule can enter

catabolic pathways and be broken down into water, carbon dioxide, and energy. If energy

intake from carbohydrates or lipids is sufficient, then excess protein will be stored as fat.

On the other hand, when energy is limiting, the body will use its own protein to provide

energy. Lean muscle tissue and other proteins can be broken down into its amino acids

constituents, deaminated, and ultimately used to provide energy.

Our body is very efficient at conserving and reusing protein, but some is lost through

the feces as unabsorbed dietary protein, through the high turnover rate of intestinal cells,

or through skin, hair and nail losses. Under normal conditions, it is only this small amount

of protein that needs to be replaced daily. The RDA for protein is 0.8 g/kg of body weight

per day.

The need for protein and amino acids is estimated by laboratory analysis but it is not

easy to measure protein in a laboratory. However, it is fairly simple to determine the

nitrogen in protein. Each amino acid contains at least one nitrogen atom and the weight of

the typical protein molecule is 6.25 times greater than nitrogen. By recording the protein

intake and dividing by 6.25, the nitrogen

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

Amino Acids and Proteins

Structure of -amino acids

The 20 Amino Acids Found in Proteins

Formation of a Peptide

Polypeptide backbone

9.bin

10.bin

Polypeptide backbone is the repeating sequence of the N-C-C-N-C-C… in the peptide bond

The side chain or R group is not part of the backbone or the peptide bond

Proteins
Make up about 15% of the cell
Have many functions in the cell
Enzymes
Structural
Transport
Motor
Storage
Signaling
Receptors
Gene regulation
Special functions

Motor- myosin
Storage- ferritin, transport- hemoglobyn
*

Importance of Proteins
Main catalysts in biochemistry: enzymes (involved in virtually every biochemical reaction)
Structural components of cells (both inside and outside of cells in tissues)
Regulatory functions (if/when a cell divides, which genes are expressed, etc.)
Carrier and transport functions (ions, small molecules)

Levels of Protein Structure
Primary Structure – amino acid sequence in a polypeptide

Secondary Structure – local spatial arrangement of a polypeptide’s backbone atoms (without regard to

side chain conformation)

Tertiary Structure – three-dimensional structure of entire polypeptide

Quaternary Structure – spatial arrangement of subunits of proteins composed of multiple polypeptides (protein complexes)

3-D Structure of Myoglobin

People with proteinuria have urine containing an abnormal amount of protein. The condition is often a sign of kidney disease.

Healthy kidneys do not allow a significant amount of protein to pass through their filters. Kidney disease often has no early symptoms. One of its first signs may be proteinuria that’s discovered by a urine test done during a routine physical exam. Blood tests will then be done to see how well the kidneys are working.

Both diabetes and high blood pressure can cause damage to the kidneys, which leads to proteinuria.

Proteinuria (Protein in Urine)
Proteinuria (Protein in Urine)

Methods of Protein Estimation
Quantitative

Biruet method
Folin-Lowry method
Kjeldahl method
Bicinchoninic acid method (BCA method)
UV method
Flourimetric method
Mass spectrometry

Protein Determination assay

Bicinchoninic Acid (BCA) method

Principle

The BCA Protein Assay is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantification 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)

Highly sensitive and selective colorimetric detection of the cuprous cation (Cu+1) using a unique reagent containing bicinchoninic acid.

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

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