Exercise 4.6 - Direct Densitometry of Gels
Figure 4.8a Model 1312 Densitometer
Figure 4.9 Protein standard scan from Model 1312
- Using the scanning cuvette supplied with your
densitometer/scanner, calibrate the scanner and choose the
proper scanning mode and wavelength filter for your
For example, for the Isco Model 1312, use the instrument on
Automatic Scan Mode with a 620 nm filter for Coomasie blue,
or a 580 nm filter for silver stained gels.
- Place the cuvette holder on the lab bench and pour about
10 ml of buffer or acetic acid into the cuvette. Unfixed
gels still in the buffer that you used to wash them will
work best for this purpose. If the gels have been fixed, be
careful of spills and minimize contact with the cuvette to
prevent corrosion of the cuvette and long term etching of
the glass plates.
- Place the stained gel into the buffer in the cuvette and
orient the gel so that the lanes are vertical as you look at
them, with the origin at the top and the front at the
bottom. Be careful to gently remove any trapped air bubbles
beneath the gel by gently lifting the gel with a blunt glass
- Very carefully lay the top glass plate onto the gel to
hold it flat. The plate should be placed on the gel at an
angle, with the two black knobs on the right, and slowly
lowered onto the gel. Again, be careful to prevent trapping
air bubbles beneath the glass plate.
- Adjust the scan rate to match that of the recorder (try
an initial scan rate of 300 cm/hr). Place the cuvette onto
the optical reading device, and manually adjust the position
of the gel so that the optical reading head of the scanner
is placed just before the origin point of the gel lane to be
scanned, in an area where there are no bands. Adjust the
baseline of the scanner and recorder to zero.
- Press and hold the START/STOP button until the scan
begins. Continue the scan until the optical head returns to
its original position. Be careful that the head does not
strike one of the knobs on the top glass plate during
operation. This will result in possible damage to the
- When all lanes are scanned, return the gel to storage
and carefully clean the gel cuvette.
- Calculate the Rf values for each peak and determine the
molecular weights of the proteins located within each peak.
Figure 4.9 presents a typical scan of protein standards.
Modern scanners are equipped with capability to output
analog data directly to various computer data management
systems, while converting the data to digital information.
In addition to storing the scan as a digital reading, these
programs can be used to integrate the area under the curves
for each peak and thereby yield quantitative data. If such a
capability is present on your system, calculate the amount
of each protein found within the major peaks.
Return to Table of Contents
Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org