Quantitative Protein Analysis/Bradford Assay

Ribosomes reading information found in mRNA are where in the cell?

Cytoplasm

Generating a complementary DNA (cDNA) from an RNA template

reverse transcription

What has naturally-occurring reverse transcriptase

Viruses?

The building blocks of proteins

Amino acids

The 4 subunits of amino acids

1. ɑ-carbon

2. Amino group

3. Carboxyl group

4. Unique R-group

What bond is formed between the a-carbon and the adjacent C and N groups

covalent bonds

What holds two amino acids together and how is bond this made? What specific molecules of the groups?

Peptide bonds are made through dehydration/condensation reaction wherein an OH from the carboxyl group and H from the amino group (water) is removed

Where is the N-terminus? what is its composition?

start of a polypeptide indicated by a free amine group (-NH₂)

Where is the C-terminus? what is its composition?

end of the polypeptide indicated by a free carboxyl group (-COOH)

List 5 ways measuring proteins are important?

1. Nutrition or consumer information

2. Bulking at the gym

3. Quantifying gene expression

4. Clinical diagnostics

5. Allergy management

What are the three means of clinical diagnostics with proteins, and what do they indicate?

1. Human C-Reactive Protein (HCRP) - indicates inflammation, maybe due to an acute or chronic condition

2. Human Serum Albumin (HSA) - the liver makes this; used in medical tests

3. Enzyme-linked immunosorbent assay (ELISA) - to identify and confirm diseases; COVID-19 detection/antigen test

Who developed the Bradford Assay

Bradford Marion

The Bradford assay is a ___ method for ___that rely on ___ with the protein

colorimetric, determining protein concentrations, CBB binding

The main reagent in the Bradford assay

Bradford reagent

Bradford reagent is mainly composed of

Coomassie Brilliant Blue (CBB) dye

Two types of CBB and how do they differ? What do we use in the laboratory?

1. R-250

2. G-250 → used in the laboratory

Differs in methyl groups

Provide the molecular formula of the CBBs and their respective MWs

1. R-250 → C₄₅H₄₄N₃NaO₇S₂; MW 825.72

2. G-250 → C₄₇H₄₈N₃NaO₇S; MW 854.02

“Concentrated Humans Never Nap On Sundays“

How does CBB bind to proteins (two ways) aka the driving forces?

1. Hydrophobic interactions

2. Van der Waals

To what amino acids does CBB bind to (chiefly and also to some extent)

• Chiefly with arginine residues

• Some with basic amino acids (histidine, lysine)

• Some with aromatic amino acids (tyrosine, tryptophan, phenylalanine)

If there is a higher concentration of proteins, what happens to the color of the solution?

It becomes more intense or brighter

What is the disadvantage with Bradford assay by itself?

Only a qualitative reading when comparing colors visually

When the solution is blue, how does light absorption work?

The blue solution absorbs its complementary color (orange) and transmits or reflects blue

What is the ideal or most optimum wavelength for absorbance of a blue color

595 nm

What do we use to measure the Bradford assay quantitatively?

UV-Vis spectrophotometer is an instrument used to compare colors and absorbance

What is the relationship between absorbance value and concentration

They are direct/linear

What are the variables of the equation of the line?

Abs = m(conc) + b

R² is also known as? What does it tell us?

It is the coefficient of determination stating how close your plots are to the regression line or how linear it is

What is a prerequisite when it comes to the spectrophotometry procedure (2)

1. Standard calibration curves must be freshly prepared because it is unstable and can easily be degraded once exposed to a solvent

2. The lamp must be warmed up first

What is the range of an acceptable R² value

0.95—1.00

What are two things that can be done is the absorbance reading is beyond the regression line or standard curve

1. Dilute the sample → lowers protein concentration

2. Add another point in the standard

What will we use as a standard for the Bradford assay?

BSA

What standard do we use for ELISA?

gamma-globulin

The interaction of the dye with the protein is ___ sensitive

pH-sensitive

What are the axes for the standard curve of the Bradford assay?

x-axis → protein concentration; y-axis → absorbance

the study of how electromagnetic radiation interacts with matter

Spectroscopy

methods of spectroscopy

Spectrophotometry

Range of visible light

370—780 nm

Range of UV light

100—400 nm

Three types of spectroscopy

1. Absorption spectroscopy

2. Emission spectroscopy

3. Scattering spectroscopy

What law is the capacity of matter to absorb light based on?

Beer-Lambert Law

Describe the Beer’s Law

↓ Intensity of transmitted light = ↑ concentration of absorbing material

Describe the Lambert’s Law

↓ Intensity of transmitted light = ↑ thickness of cuvette/path length

Equation of the Beer-Lambert Law

A = log(I₀/I)= εcl

Incident light?

source to sample

Transmitted light?

passes through the sample toward the detector; light unabsorbed by the sample

If the sample has more volume, then ___

more molecules can absorb the light

Path length?

volume of the sample, the thickness or size of the cuvette, and the intensity of color inside the cuvette

Transmittance is inversely proportional to? (2)

1. Absorbance

2. Path length

Absorbance is directly proportional to? (2)

1. concentration

2. path length/thickness of absorbing material

Which CBB variant has methyl groups?

G-250

What are the three forms of G-250

1. Cationic (reddish tint) → 470 nm

2. Neutral (greenish tint) → 650 nm

3. Anionic (bluish tint) → 595 nm

“Red Cars Zoom 470, Green Nissans 650, Blue Airplanes 595”

Under acidic conditions, the dye is predominantly in what form?

doubly-protonated, red cationic form

Proteins are generally what charge?

Neutral

What charge do proteins have in acidic environments?

Positively charged

Before reacting with proteins, the Bradford reagent is in what form and has what tint/color?

cationic, unstable form with a brownish-red tint

What happens with CBB is introduced to a protein?

CBB can donate a lone pair of electrons to the protein’s ionizable groups (aka their side chain/R group)

When the protein receives the CBB’s lone pair, it becomes?

unstable

In what manner does CBB bind to the protein under acidic conditions (2)

1. hydrophobic interactions

2. Van der Waals interaction

For CBB to bind stably to protein, it needs to be?

doubly protonated

Describe what happens for hydrophobic interactions?

• When dye comes in contact with protein, it results in exposure of hydrophobic pockets of the protein

• If this happens, CBB will take advantage as it can bind to the hydrophobic pockets, thus hydrophobic interactions

• CBB Dye ⇔ protein = exposed hydrophobic pockets of the protein

Van der Waals are ___-dependent interaction between ___ molecules and is relatively ___ than ___ or ___ bonds that's why CBB binds ___ to the protein

distance-dependent, charged, stronger than ionic or covalent bonds, stably

The ___ groups of CBB bind to ___ of amino acids

Sulfonic groups of CBB bind to positive amines of amino acids

What does the binding of CBB and protein create?

Protein-dye complex

Summarize the two interactions of CBB and protein

1. CBB (sulfonic group) + amino acid (amino group) = Van der Waals

2. CBB + hydrophobic pockets = Hydrophobic interactions

Why are hydrophobic pockets created?

Because CBB shares electrons, exposing hydrophobic bonds

The creation of interactions happens ___

simultaneously

What are 6 criteria to consider with the Bradford assay standard curve?

1. Acceptable R2 value

2. Distance of the points → there must be sufficient difference between the absorbances

3. Gradient colors

4. Increasing color intensity

5. Absorbances must be increasing

6. Points must fall close to the regression line

How do we avoid bad/undesirable results for our standard curve?

Accurate and precise pipetting

What is the proper formula for determining protein concentration from the equation of the line

Abs = m(conc) + b * DF

What is an important variable we have to account for when sometimes calculating for the protein concentration

dilution factor

What is the formula for the dilution factor?

DF = final volume of sample (addition)/initial volume of sample

When do we add the Bradford reagent?

Added last and at the same time for each of the samples/cuvettes