BIO 220 Laboratory Exercises
Introduction to Laboratory Techniques
This exercise consists of three parts:
1) Use of a pH meter and preparation of homogenization media
2) Use of the UV-VIS spectrophotometer
3) Preparation of a standard curve for protein quantitation
These three tasks are commonly performed in cell and molecular biology research laboratories.
Part 1. Use of a pH meter and preparation of homogenization media
Upon completion of this exercise, students will be able to:
- Standardize and use a pH meter
- Prepare a biological buffer and adjust the pH
In the next lab exercise, mitochondria will be isolated from rat liver using
homogenization media during disruption of the cells. A goal of today's exercise is to prepare this
solution. The media contains 0.25 M sucrose, 1 mM ethylenediaminetetraacetic acid (EDTA),
and 50 mM Tris-HCl, pH 7.4. Each group of 3 students must prepare 200 mL of homogenization
media for use at the next lab meeting.
The pH meter is essentially a voltmeter designed to measure the potential difference
between a reference electrode and a sensing electrode. The reference electrode usually contains
a solution of AgCl2, and the solution to be tested contains varying amounts of protons (H+). The
instrument measures the difference in electrical potential (mV) between these solutions and
translates this into a pH reading. Obtaining an accurate reading with a pH meter depends on
effective standardization, static charge, solution temperature, and other factors. Generally, it is
desirable to calibrate the meter with a standard buffer of a pH as close as possible to the pH of
the solution to be measured. The problem of static charge can be minimized by blotting, not
wiping, the electrode gently with a Kimwipe before transferring to a new solution. The
temperature of the calibration solution should be as close as possible to the desired working
temperature of the solution to be tested.
Safety Notice
Concentrated solutions of acids and bases will irritate eyes and skin. Be careful not to get these
solutions in your eyes or on your skin.
Procedure
1) Standardize the pH meter with pH 7 buffer.
2) Weigh the correct amount of Tris-base or Tris-HCl (M.W. = 121.1 g/mol) to make 200 mL of
50 mM buffer. Weigh proper amounts of sucrose (M.W. = 342.2 g/mol) and EDTA (M.W. =
372.2 g/mol) to yield final concentrations of 0.25 M and 1 mM, respectively, in 200 mL.
3) Dissolve the solids in approximately 150 mL of distilled water. This allows room for
addition of acid or base to adjust the pH. Record the initial pH of the solution and titrate
to pH 7.4 with either 1 M HCl or 1 M NaOH, as needed.
4) Transfer the solution to a graduated cylinder. Add distilled water for a final volume of
200 mL. You may wish to recheck the pH of the solution.
Part 2. Use of the UV-VIS spectrophotometer
Upon completion of this exercise, students will be able to:
- Properly use variable-volume micropipettors
- Prepare simple dilutions of a stock solution (i.e., 1/5, 1/10, etc.)
- Use the UV-VIS spectrophotometer to record absorbance values
- Generate a graph for determination of an extinction coefficient ()
The colorimeter (spectrophotometer) is an instrument that measures the amount of light
absorbed by molecules in solution. The specimen is placed in a special glass or plastic cuvette.
Light of a given wavelength is passed through the sample, and exiting light is measured by a
phototube (detector). These machines automatically display the amount of light absorbed as
absorbance (optical density) values. Absorbance is a unitless quantity, and is calculated by the
following relationship:
Where A= absorbance, Io = intensity of light entering the cuvette, and IT = intensity of light
exiting the cuvette and reaching the detector.
A number of factors will affect the light absorbed by a given substance including:
concentration, the solvent used, the path length, and the characteristics of the cuvette. It is
possible to determine the absorbance of a substance and calculate the absorptivity coefficient ().
These coefficients are useful because they can be used to determine the concentration of a
substance under defined conditions. The equation used to determine concentration is call the
Beer-Lambert equation:
Where l = path length, and c= concentration. In this lab, you will lean how to use the
spectrophotometer, test your ability to accurately dilute a dye, and determine the absorptivity
coefficient (, also called the extinction coefficient) of dichlorophenolindophenol (DCIP), an
electron acceptor that we will use in future laboratory periods.
Procedure
1) Turn on the spectrophotometer using the switch on the back right hand side. Make sure the
instrument has been on at least 10 min before use.
2) Set the wavelength to 600 nm by using the keypad and "Go to ".
3) Prepare 4 dilutions of 0.5 mM dichlorophenolindophenol (DCIP) dye: 1/5, 1/10, 1/50, and
1/100. The dilutions may be prepared directly in plastic cuvettes (3 mL total volume). Use
distilled water as the diluent. Prepare an additional "blank" cuvette that contains only water.
4) Zero the spectrophotometer.
There are different methods available for "zeroing", "blanking", or "background-correcting" a
spectrophotometer. In this course, an air-air background correction will always be used.
Make sure there are NO CUVETTES in the instrument. Close the cover. Check that
the instrument is set at the desired wavelength. Press the "Auto Zero" key. Once the display
reads "0.000", the instrument is ready to use.
5) Place the blank cuvette containing water in the sample holder towards the back of the
instrument, and one diluted dye sample in the sample holder in the front. Close the lid and
record the absorbance at 600 nm.
6) Measure the absorbance (600 nm) for every diluted sample. Construct a graph of
concentration versus absorbance. If your dilutions are perfect, a linear relationship will result.
Determine "", the slope, from the graph.
Part 3. Preparation of a standard curve for protein quantitation
Upon completion of this exercise, students will be able to:
- Dilute bovine serum albumin (BSA) for use as a standard in protein quantitation
- Prepare a standard curve for the Bio-Rad Protein Assay, using BSA as the standard
- Graph data for the standard curve, and observe the linear relationship between amount of
protein and A595 nm.
The Bio-Rad Protein Assay is a dye-binding assay based on the differential color change
of the dye in response to different protein concentrations. This is a colorimetric assay, and the
amount of protein in the unknown sample is determined by comparing the absorbance of the
unknown sample to the concentration-versus-absorbance relationship defined by the standard
curve. The assay uses Coomassie Brilliant Blue G-250 dye, and is based on a method developed
by Dr. Marion Bradford at the University of Georgia1.
The Bio-Rad assay is linear between 20 and 140 µg protein in a 0.1 mL sample. A
sample standard curve, as reproduced from the technical literature for the Bio-Rad Protein
Standard Assay2, is shown in Figure 1. The standard curve and sample points should be prepared
at the same time. Absorbance readings (A595 nm) must be recorded 5 min to 1 hr after addition
of the dye reagent. A new sample curve must be prepared each time the assay is run.
Figure 1. This is an example of a Bio-Rad Protein Assay standard
curve, reproduced from the technical literature2.
Safety Notice
Bio-Rad Dye contains phosphoric acid, and will stain skin and clothing. Avoid getting the dye in
your eyes, on your skin, or on your clothes.
Procedure
1) Each group will be provided with 200 µL of a 1.4 mg/mL BSA standard. This is enough
material to complete the exercise, provided that good pipetting technique is used.
2) Prepare the standard curve using clean, dry, glass test tubes.
3) Make a table showing how much BSA standard and water to combine to make 0.1 mL of the
following concentrations of BSA: 140, 70, 35, 17.5, and 0 µg/µL.
4) Following the table prepared in step 3, add the proper amounts of water and BSA standard to
each glass tube. Important: Use a fresh tip for each transfer of BSA.
5) Use the bottle-top dispenser to add 5 mL of Bio-Rad Dye to each tube. Place a small piece of
Parafilm over the tube, and vortex to mix.
6) Transfer (by pouring) solutions into plastic disposable cuvettes. Make sure cuvettes are filled
to the mark.
7) Use the spectrophotometer to record the absorbance at 595 nm. The cuvette containing
0 µg/µL BSA should be used as the reference.
References
1) Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein
utilizing the principle of protein dye-binding. Anal. Biochem. 72, 248-254.
2) Bio-Rad Laboratories, Hercules, CA. (1999) Instruction Manual, Bio-Rad Protein Assay, Rev C., Bulletin
Number LIT33, p. 5.