33 Identification of Cell Lines
329
1. Apply the sample (15 fll) containing 12 flg protein to a fllter paper (LKB Electrofocusing
Instruments).
2. Place the filter paper on the gel plate approximately 22 mm from the
cathode strip of the plate.
3. Run the focusing at 30 W, 50 rnA and 1,500 V across the width of the
gel for 15 h.
4. Remove the fllter paper on which the sample was applied 30 min after
the start of the procedure by stopping the focusing, and restart the
focusing after removal of the fllter.
5. After the focusing is completed, measure the pH of the gel surface
using a surface electrode and pH meter.
6. Refocus the gel for another 15 min.
1. Place the gel plates in a shallow tray, and overlay them with staining Staining
solution.
2. Develop the bands in the gel for 10-15 min at room temperature.
3. Wash the gel in cold tap water for 2-5 min.
Results
According to McIntosh and Ignoffo (1983), isoelectric focusing using the
enzyme phosphoglucoisomerase differentiated between cell lines from
different species as well as between cell lines of the same species. For
example the isoelectric point values (pI) of the major bands were 5.35
for the Heliothis zea cell line, 5.35, 5.82, and 6.82 for the Heliothis armigera
cell line, 4.84, 5.10, and 5.35 for the Heliothis virescens cell line, 5.28 for
the Plutella xylostella cell line, and 5.00 for the Spodoptera frugiperda
cell line (McIntosh and Ignoffo, 1989).
Subprotocol 3
DNA Amplification Fingerprinting (DAF)
McIntosh et al. (1996) applied PCR amplification of mammalian aldolase, prolactin receptor, and interleukin (lL)-l /3 to distinguish insect cell
lines.
Materials
- Thermal cycler
- UV illuminator
- Electrophoretic apparatus
- Polaroid camera (Polaroid Corporation)
- Physiological saline
Equipment
Solutions
329
1. Apply the sample (15 fll) containing 12 flg protein to a fllter paper (LKB Electrofocusing
Instruments).
2. Place the filter paper on the gel plate approximately 22 mm from the
cathode strip of the plate.
3. Run the focusing at 30 W, 50 rnA and 1,500 V across the width of the
gel for 15 h.
4. Remove the fllter paper on which the sample was applied 30 min after
the start of the procedure by stopping the focusing, and restart the
focusing after removal of the fllter.
5. After the focusing is completed, measure the pH of the gel surface
using a surface electrode and pH meter.
6. Refocus the gel for another 15 min.
1. Place the gel plates in a shallow tray, and overlay them with staining Staining
solution.
2. Develop the bands in the gel for 10-15 min at room temperature.
3. Wash the gel in cold tap water for 2-5 min.
Results
According to McIntosh and Ignoffo (1983), isoelectric focusing using the
enzyme phosphoglucoisomerase differentiated between cell lines from
different species as well as between cell lines of the same species. For
example the isoelectric point values (pI) of the major bands were 5.35
for the Heliothis zea cell line, 5.35, 5.82, and 6.82 for the Heliothis armigera
cell line, 4.84, 5.10, and 5.35 for the Heliothis virescens cell line, 5.28 for
the Plutella xylostella cell line, and 5.00 for the Spodoptera frugiperda
cell line (McIntosh and Ignoffo, 1989).
Subprotocol 3
DNA Amplification Fingerprinting (DAF)
McIntosh et al. (1996) applied PCR amplification of mammalian aldolase, prolactin receptor, and interleukin (lL)-l /3 to distinguish insect cell
lines.
Materials
- Thermal cycler
- UV illuminator
- Electrophoretic apparatus
- Polaroid camera (Polaroid Corporation)
- Physiological saline
Equipment
Solutions