BIOL 395 First Exercise

Determination of A Standard Curve for Protein Concentration

The purpose of this exercise is to allow you to become familiar with some of the laboratory
equipment you will be using throughout this course, and the concept and use of a standard curve.
You will test your skill in using the P-20 and P-200 micropipettes by constructing a standard curve
for determination of the concentration of an unknown protein. If you use the pipettes correctly, the
curve that you generate by plotting absorbance vs. concentration of an unknown protein should be a
straight line.
Protein concentrations will be determined using the method of Bradford. The Bradford procedure
is a dye-binding assay based on the differential color change of Commassie blue G dye as it binds to
protein. In both research and clinical applications, this assay has replaced earlier procedures for
protein measurement because of its simplicity, the stability of the color change, and the lack of
interference from non-protein components.
The Bradford assay is based on the observation that the absorbance maximum for an acidic
solution of the dye shifts from 465 nm to 595 nm when binding to protein occurs. Within a range of
5-100 ug of protein, a linear relationship exists between protein concentration and the increase in
absorbance of the dye solution at 595 nm. Over a broader range of protein concentration, the dye
binding method gives an accurate, but not entirely linear response.
Procedure:
1. The instructor will provide a solution of a purified protein, bovine serum albumin (BSA), at a
known concentration (10 mg/ml). Make a series of dilutions of the BSA standard solution (10
mg/ml) into 1.5 ml microcentrifuge tubes to give final concentrations of 0.25, 0.5, 0.75, 1.0, and
1.25 mg/ml of protein. Make 0.1 ml (100 ul) of each dilution. As a diluent, use distilled water.
Mix thoroughly.
2. Place 20 ul of each dilution into individual microcentrifuge tubes and make up an additional tube
containing 20 ul of water for use as a reagent blank. Add 1 ml of diluted Bradford dye reagent to
each tube (6), close the tube, and vortex. After at least 5 min, but before 1 hr, transfer to a plastic
cuvette. Use the reagent blank to zero the spectrophotometer at 595 nm, then read the absorbance
of each sample at 595 nm. Record the absorbance of each sample in your notebook.
3. Standard curve. Plot, either on graph paper or on the computer using the program Excel, the
absorbance of each sample vs. the protein concentration.
Note that the Bradford assay is subject to variation from day-to-day, and from one batch of dye
reagent to another. Therefore, it is necessary to construct a new standard curve each day, or when
a different bottle of reagent is used.

Extra Credit
(1% of the overall course grade)
Bring to the next laboratory session a fully labeled graph of your standard curve. You do not need to
submit it to turnitin.com, but it should be your own work. Points will be awarded as follows:
2pts Detailed title, i.e. a full sentence that summarizes the graph.
3pts Detailed legend that provides sufficient information for the reader to understand what has
been done. Usually 2-3 sentences is sufficient.
2pt Labeled x and y axes including units of measurement (absorbance does not have true units).
1pt Data points plotted.
1pt Best fit line added.
1pt If an unknown sample had 0.4mg/ml of protein in it, what Absorbance at 595nm would you
have obtained in your experiment?
Please refer to the Figures Examples file in the Resources folder on WebCampus for examples.
Purpose of the exercise
Scientific papers consist of text, figures and tables. Figures are frequently the most important part of
the paper as they present the data being reported. In our scoring schemes, figures are assigned point
values accordingly. Students frequently lose significant numbers of points due to insufficient detail
and labeling. A useful check is to examine whether a reader could understand the figure from the
title and legend without reference to the text. The aim is to allow the reader to understand the data,
and less emphasis on how it was obtained to avoid too much repetition of materials and methods.
For future reference:
*Graphs are figures in scientific papers.
*In a write up, you title will be preceded with the figure number.
*Each figure should have a unique title. For example, if a report has more than one standard curve, it
should be easy for the reader to distinguish between them. Avoid confusion. Avoid titling figures
Gel Image, but rather state what samples are on the gel.
*By convention, the known variable (e.g. concentrations or sizes of standards) is put on the x-axis,
and the unknown or determined variable (e.g. absorbance, distance migrated) is put on the y-axis.
*Unknown sample readings should not be part of the standard curve!