328
Related Techniques
Subprotocol 2
Phosphoglucoisomerase Isozyme Pattern on Isoelectric
Focusing
Mcintosh and Ignoffo (1989) developed a method to identify insect cell
lines by comparing isozyme patterns obtained on isoelectric focusing.
Materials
Equipment - French press (American Ins.)
- Centrifuge and centrifuge tubes (e.g. Eppendorf Microfuge; Brinkman
Institute)
- Electrofocusing apparatus
- Surface electrode (LKB Instruments)
- pH meter ( 71 pH m Beckman)
- Ultra low-temperature freezer
- Ampholine polyacrylamide gel plate pH 3.5-9.5 (LKB Instruments)
- Filter paper (0.5 x 1.0 cm; LKB Instruments)
Solutions - Buffered saline
Preparation
of cells
Preparation
of enzyme
- Staining solution for phosphoglucoisomerase (30 ml of 0.6 M Tris,
pH 8.0, 76 mg sodium EDTA, 120 mg MgCI 2 ·6H 2 0, 20 mg fructose-6phosphate, 25 IV glucose-6-phosphate dehydrogenese, 12 mg 3-(4,5dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), 6 mg
NADP (nicotinamide adenine dinucleotide phosphate, 12 mg phenazine methosulfate, 30 ml of 2% (w/v) agar in H 2 0) (Brown and
Knudson, 1980)
Procedure
1. Collect the cells to be analysed at the logarithmic growth phase by
dispersing the cells in the medium and centrifuging (150 gfor 5 min)
the resulting cell suspension.
2. Harvest approximately between 10 7 and 10 8 cells, wash them three times
with Ca 2 +- and Mg2+-free buffered saline, and suspend the cells in the
same buffer solution.
1. Apply a pressure of 10,350 kPa to the cell suspension using a French
press (if a French press is not available, break the cell membranes by
freeze-thawing, or by using a cell disintegrator).
2. Centrifuge the suspension at 15,600 g for 3 min. If necessary, determine the protein content at this stage (e.g., the Bio-Rad Protein Assay
Kit II (Bio-Red) may be convenient).
3. Store the sample at -70°C until electrofocusing.
Related Techniques
Subprotocol 2
Phosphoglucoisomerase Isozyme Pattern on Isoelectric
Focusing
Mcintosh and Ignoffo (1989) developed a method to identify insect cell
lines by comparing isozyme patterns obtained on isoelectric focusing.
Materials
Equipment - French press (American Ins.)
- Centrifuge and centrifuge tubes (e.g. Eppendorf Microfuge; Brinkman
Institute)
- Electrofocusing apparatus
- Surface electrode (LKB Instruments)
- pH meter ( 71 pH m Beckman)
- Ultra low-temperature freezer
- Ampholine polyacrylamide gel plate pH 3.5-9.5 (LKB Instruments)
- Filter paper (0.5 x 1.0 cm; LKB Instruments)
Solutions - Buffered saline
Preparation
of cells
Preparation
of enzyme
- Staining solution for phosphoglucoisomerase (30 ml of 0.6 M Tris,
pH 8.0, 76 mg sodium EDTA, 120 mg MgCI 2 ·6H 2 0, 20 mg fructose-6phosphate, 25 IV glucose-6-phosphate dehydrogenese, 12 mg 3-(4,5dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), 6 mg
NADP (nicotinamide adenine dinucleotide phosphate, 12 mg phenazine methosulfate, 30 ml of 2% (w/v) agar in H 2 0) (Brown and
Knudson, 1980)
Procedure
1. Collect the cells to be analysed at the logarithmic growth phase by
dispersing the cells in the medium and centrifuging (150 gfor 5 min)
the resulting cell suspension.
2. Harvest approximately between 10 7 and 10 8 cells, wash them three times
with Ca 2 +- and Mg2+-free buffered saline, and suspend the cells in the
same buffer solution.
1. Apply a pressure of 10,350 kPa to the cell suspension using a French
press (if a French press is not available, break the cell membranes by
freeze-thawing, or by using a cell disintegrator).
2. Centrifuge the suspension at 15,600 g for 3 min. If necessary, determine the protein content at this stage (e.g., the Bio-Rad Protein Assay
Kit II (Bio-Red) may be convenient).
3. Store the sample at -70°C until electrofocusing.