Module 1: Bradford Assay : Determining protein concentration

Bradford Assay

Based on the formation of a complex dye (Coomassie blue) and proteins in solution

- As protein bind the dye, there is a change from brown to blue.

- The absorption spectrum can be recorded using a spectrophotometer at a wave length of 595 nm

Reason to conduct a protein assay

During a purification of a protein, you need to know how pure your sample is by determining the amount of enzymatic activity vs protein concentration

Protein Assay

Method of detecting for the presence of a specific protein and estimating the concentration of the protein

The absorption is proportional to the amount of dye bounded

hence the amount of protein in the solution the absorption can be compared to a solution of known protein, BSA ( Bovine Serum Albumin ) concentration

A standard curve is fitted through the data points and the absorbance of the unknown protein

is then matched with the respective concentration in the curve

- absorbance of different BSA concentrations are measured and plotted

Light Spectroscopy

A spectrophotometer separated white light into a spectrum of colors ( wavelength) and measures the amount of light absorbed by a dissolved chemical

How to use a spectrophotometer

1. Clean the cuvette with a kim wipe

2. turn on the spectrophotometer 10-15 minutes before lab

3. place a blank (a cuvette that contains only the solvent used to dissolve the chemical you are analyzing)

4. adjust the filter to the lowest wavelength and then adjust the wavelength until it is 0

5. remove the blank and add a cuvette with the chemical and solvent

6. continue and remember to put in the blank to make sure it is 0

7. adjust wavelength if needed

Light passes through the cuvette and measures the pattern of absorbance and transmittance

then, we can use this information to identify the concentration of the sample

- light is either absorbed by the dissolved substance or transmitted through the solution and exits the sample tube

The absorbance has a logarithmic relationship to the transmittance

An absorbance of 0 corresponds to a transmittance of 100% (1).

Beer-Lambert Law

law stating that intensity of color change is directly proportional to the concentration of an analyte in a solution

More solute, the lower the transmittance

more solute, higher absorbance

Which button on the spectrophotometer machine is used to set the blank?

0 ABS 100%T

When using frosted or ridged cuvettes, how should you handle the sample cuvettes (which side should you touch with your fingers, and which should you avoid)?

avoid the clear side

How to place cuvette in the chamber?

the arrow on the front of the cuvette should be oriented in the direction of the light path.

Serial Dilution

Dilution of a substance several times by the same amount each time

Dilution

the process of decreasing the concentration of a solute in a solution

- this can be done by simply by mixing it with more solvent like adding more water

Dilution are useful when we want to obtain a very diluted solution from very concentrated stock solution

C1V1=C2V2

By using serial dilution, we first look at the final volume and concentration needed, then determine the dilution factor (DF)

Dilution Factor = final concentration / initial concentration

Dilution Factor (DF)

final concentration / initiation concentration

Standard Curve Graph

a plot of absorbance vs. a varying amount of some known concentration of protein

You have been given a stock solution of dye that has a 10X concentration. You wish to make 870ul of a 1X dilution of this dye. Give the volume of water that you will use (in ul)

C1V1= C2V2 ; V1 = (C2V2)/C1 ; V1 = (870(1)/10) V1=87ul. ; 870ul-87ul = 783ul of water

You have been given a stock solution of dye that has a 10X concentration. You wish to make 750ul of a 1X dilution of this dye. Give the volume of 10X dye that you will use (in ul)

C1V1=C2V2 ; V1= (C2V2)/C1 ; V1= (750(1)/10) V1= 75ul

What dye is used to measure the concentration of proteins in the Bradford Assay we will perform?

Coomassie blue

You have a stock solution of protein that you would like to dilute. How much stock protein do you need to add to 800 μL of buffer to make a 1:5 dilution?

(stock/stock+buffer)=1/5 ; x=200

When measuring absorbance of a sample, what does the blank contain?

the solvent

To read the absorbance of a sample, what button must you press?

None, the reading is automatic

When loading the cuvette in the chamber of the spectrophotometer, the arrow on the cuvette should face the direction of the light

True

In a Bradford Assay, when absorbance increases, the protein concentration decreases

False : more solute more absorbance

Why is it important to dilute samples when performing a Bradford Assay?

Bradford works at low concentration. You need a sample concentration that falls within the range of measurable concentrations)

The coomassie dye we added when performing the Bradford assay stains proteins. How does it color change with increasing protein concentration?

the blue color intensifies