and Jessie Brantwein
1993 Woodrow Wilson Biology Institute
Bio Rad Assay
Bio Rad Assay: The biotechnology connection with
this assay is mainly to learn the
instrumentation procedures for the spectrophotometer
and the micropipette both of which
are invaluable tools in modern research. The graphing
of the spectrophometer readings or any quantitative results is
an indispensible tool which students must learn early in the AP
and general Biology course.
PAGE Gels: Electrophoretic procedures, either vertical
acrylamide or horizontal agarose systems, are increasingly being
utilized for a variety of diagnostic, research and investigatory
procedures. Again, graphing results against a standard is a valuable
When conducting laboratory assays for organic compounds, most Biology classes utilize a very simple protein test called the Biuret method. When combined with the protein in question, the blue Biuret reagent becomes a pale violet. However, this test does not indicate protein concentration or molecular weights.
One method for determining concentration is the Bio Rad Protein Assay which is a dye-binding procedure responding to various concentrations of soluble protein containing tyrosine, tryptophan or arginine resudes. Dilutions of the protein sample are made with a buffer such as Tris, and the Tris is used as a blank. The completed reaction is read on a spectrophotometer such as a "Spec 20" at 595 nm wavelength. Samples are plotted against a standard such as bovine serum albumin or bovine gamma globulin. A standard curve is set up plotting optical density against protein in µg/µl. For further information on this assay, one may investigate the Lowry (Folin-Ciolcalteau) or Bradford methods.
Molecular weights of unknown proteins may also be
determined by the use of SDS polyacrylamide gel electrophoresis.
When samples are run on "PAGE" gels, varying mobilities
of proteins are due to the sieving effect of the gel. The ratio
of acrylamide to bis-acrylamide sets up the size of the pores
in the gel. One may run native gels with the protein intact,
or one may denature with SDS, heat and _-mercaptoethanol. When
the mobility of the sample proteins is plotted against a standard
ladder, one may read the molecular weight in kiloDaltons. A ratio
may be set up according to the mm's of mobility against the standard.
Assay may be used in general and more advanced biology and chemistry
PAGE gels are probably geared more towards the AP
Biology and Chemistry class, but the graphing against standard
measures could prove beneficial to general courses.
Students: The Bio Rad reagent is rather inexpensive since it must be mixed on 5:l ratio. If students are placed in groups it is advisable to put no more than two or three to a group or there is a tendency for some of the students to observe only. This procedure should take no more than one laboratory period if all members of the group are participating: one for dilutions and one for reading the spectrophotometer .
The PAGE gels should be cast by the teacher. Therefore,
the protein preparation (denaturing) and dilutions may be completed
by each group. It will take approximately two periods to complete
this laboratory if the gels are previously cast. One period
will be needed to denature the protein and one for running of
the gel. The teacher may elect to stain with the Coomassie Blue.
Bio Rad Materials:
PAGE Gel Materials:
All solutions may be diluted
and disposed of down the sink.
Student: Student should
wear goggles and gloves for both the Bio Rad and PAGE gel procedures.
Students should not cast gels; only deposit the samples in the
Teacher: For the Bio
Rad Assay, wear goggles and gloves. For the PAGE gel preparation, presence of a carcinogen and mutagen in acrylamide
requires gloves, mask and goggles.
Bio Rad: Time required:
20 minutes to dilute and filter the concentrate
| Solutions: 30 mls of Dye
Tris Solution: (50 mM)|
PAGE Gel Solution: (Allow several hours for solution
preparation and at least one hour to cast the gels. The gel will take several hours to run and several hours to destain.)
Lower (Resolving) Gel
For Two Gels|
(For 15 ml mixture)
Upper (Stacking) Gel
(For 10 ml mixture)|
For 500 ml:|
For 4X Buffer:|
Preparation of Sample:|
Gel "Fixing" Solution:
Coomassie Brilliant Blue Stain|
Destain for 2 hours (several hours)
|Most solutions will keep for several months if refrigerated and sealed properly.|
Bio Rad Assay:
(See hard copy for "Dilution Aliquots"
(See hard copy for Page Gel Simulation)
protein2: Spectrophotometric Readings
(See "Results" Graphs on Hard Copy)
Ausubel, F. M., et. al. Current Protocols in Molecular Biology,
Volumes 1 and 2, John Wiley and Sons, New York, 1989.
Ausubel, F. M., et. al. Short protocols in Molecular Biology,
John Wiley and Sons, 1989.
Bio Rad Protein Assay
Bio Rad Life Sciences Group
2000 Alfred Nobel Drive
Hercules, Ca. 94547
Clark, John M. Jr., and Robert L. Switzer, et al. Experimental
Biochemistry ,(2nd ed.) W. H. Freeman and Company, New
Slater, Robert J., et al. Experiments in Molecular Biology,
Humana Press, Clifton, New Jersey, 1986.