Sunday, January 16, 2011

Bradford Protein Assay~

Happy LATE New Years~ I am sorry, I been busy studying for school (finals is coming up) and organizing data from the lab into my notebook. However.....

Recently, in my lab at CalTech, I did a lab on Bradford protein assay.  It is a procedure based on an absorbance shift of the Coomassie Brilliant Blue G250 dye.  When the dye binds with proteins, it convert from its original red form into a bluer form.  The bound form of the dye has an absorption spectrum maximum held at 595 nm.  Certain concentration of detergents, which is used to lyse cells, interferes with the Bradford assay.

I used Bovine Serum Albumin (BSA) as a known protein to create a calibration graph of concentration versus absorbance.

Prepare BSA solutions:

I was given BSA solution of 1 ug/ml, and I need to prepare BSA concentrates at 2 ug/ml, 5 ug/ml, 7 ug/ml, 10 ug/ml, 20 ug/ml, and 30 ug/ml.

Concentration (ug/ml)
BSA solution (based on 1 ug/ml)
Bradford reagent dye
2 ul
998 ul
5 ul
995 ul
7 ul
993 ul
10 ul
990 ul
20 ul
980 ul
30 ul
970 ul

All of the solutions have the final volume of 1000 ul or 1 ml.

I, then, input 2 ul of the solution into the nanometer, which has the Bradford protein assay procedure pre installed into the computer.

^ Instrument I used.

^The display.

A calibration graph is formed on the concentration versus absorbance.  The graph uses the Beer-Lambert Law:
(where Eb are constant, sometimes written as k and c as the concentration)

The calibration graph look something like this:

Notice the r squared variable.  In statistics, it is known as the coefficient of determination.  It determines the linearity of the graph.  In other words, the closer the variable is to 1, the more linear it is.  

Using the calibration graph, I was able to determine the concentration of several biological samples based on the absorbance of the samples and the corresponding absorbance over concentration.  

The following are a list of biological samples, which I performed the Bradford assay on in order to find the concentration:

Possible sources of error:
I performed the calibration graph and the actual experiment on two different days, so the results are not as accurate as they possibly are.
There are some samples with 0 concentration.  One thing is that I did not sanitize the nanometer correctly or air went into the machine while the experiment is performed.

This conclude the experiment. XD


  1. Good job,

    you can also quite nicely see that Lambert-Beer's law is only valid for dilute concentrations as you notice some saturation towards the higher concentrations.

    Your individual measurements are close together. That means that you have good pipetting skills!

  2. Hi!
    I am performing the same assay and I got confused with dilutions. I didn't dilute my sample (I used the protocol of Bradford 1976) using an aliquot of 0.1 mL. Now, when I am transforming my data, do I need to take in count the total volume of my sample? in this case, I am working with fasting urine samples.

    Thanks in advance!