ISOENZYME ANALYSIS

LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

REFERENCE NO: AHC/1998/3/3.1/2.2

TITLE: ISOENZYME ANALYSIS

INTRODUCTION

Isoenzyme analysis is based on the existence of enzymes with similar or identical specificity, but different molecular structure isoenzymes). Isoenzyme analysis is used to study the patterns of migration of isoenzymes present in cell lysates following electrophoresis using agarose gels. The patterns obtained are species specific and therefore are used as quality control and authentication procedures to confirm species of origin of material.

PROCEDURE

Preparation

Set Sorvall RT6000 centrifuge at 4°C.
Place a bottle of PBS at 2 to 8°C.
Place a bottle of trypsin/EDTA at 37 ± 2°C if attached lines are to be tested.
Label 2 cryotubes for each sample with cell name, CB number and date and place in rack at <-10°C.
Label a 50ml tube for each sample with cell name (more than one may be needed for a suspension line).
Label a 15ml tube for each sample with the cell name and place on ice.

Extraction
Attached Cells

Attached Cells

From a confluent 75cm² flask decant approximately 30ml of media into the labelled 50ml tube. Discard any remaining media.
Wash cells by pipeting 10ml of PBS into flask. Gently rotate and then decant PBS. Repeat with 10ml fresh PBS.
Add 2ml of trypsin to sample, and draw off the excess leaving approximately 0.5ml.
Incubate at 37 ± 2°C for up to 5 minutes, or until cells detach.
When cells have detached, harvest them into the 50ml centrifuge tube containing the original medium.

Suspension Lines

From a confluent 75cm² flask decant all cells and media into 50ml tube(s).

Enzyme Extraction

Centrifuge cells at 150g for 5 min and a temperature of 2 to 8°C.
[Transfer tubes onto ice] Decant the medium to waste, resuspend the pellet(s) in a small volume 1 to 2 ml of ice cold PBS and recombine if there is more than one per line. Make up to 10ml with cold PBS. Transfer to a labelled 15ml tube.
Centrifuge as above.
Decant the PBS and drain the 15ml tube onto absorbent paper to remove any excess. With a swab or rolled up tissue paper absorb the rest of the PBS from the pellet.
Add a volume of cold extraction buffer equivalent to the size of the pellet (approximately 100ml) and mix well.
Leave tubes on ice for 10 to 15 min.
Check that the cells have lysed by taking a small sample and placing on a microscope slide. Cover with a coverslip and view using a microscope. If lysis is incomplete leave on the ice for a few minutes longer.
Centrifuge the extract at 1900g for 5 min at 2 to 8°C.
Transfer 20ml of clarified supernatant to each of the pre-labelled cryotubes and keep on ice. Snap freeze 1 of the 2 samples in liquid nitrogen.

N.B. Do not keep samples on ice for longer than is necessary, i.e. up to 1 hour.

Running the Gel

Pour 90ml of electrophoresis buffer (Authenti-Kit or equivalent) into each well of bottom unit of the electrophoresis chamber - no bubbles.

Remove the hard plastic backing from the gel (Authenti-Kit). Normally 2 gels are prepared, one for each enzyme e.g. G6PD and LD.

Use a Hamilton micropipetter to load 1ml of each sample into different wells, using a separate applicator tip for each sample.

Load the standard, L929, into the first well, the control, Hela S3, into the second well and the samples into the remaining wells. Ensure that the same sequence is used in each gel.

Place the gel in the upper unit of the electrophoresis chamber with the '+' signs aligned on the gel and chamber.

Place the upper unit over the bottom unit and insert the supply leads to the lower chamber first then to the powerpack supply.

N.B. Red ® + Black ® -

Gradually turn the voltage up to 160v (constant voltage) and electrophorese for 30 min.

After 20 min of electrophoresis, make up the enzyme reagents by reconstituting the lyophilized enzyme substrates (Authenti-Kit reagent) with 0.5ml of sterile distilled water. Record batch numbers.

After 30 min of electrophoresis, remove the gels from the electrophoresis chamber. Add one enzyme reagent to one each of the gels and distribute evenly over the gel surface with a pasteur pipette.

Place the gels in separate black developing trays and incubate at 37 ± 2°C.

Check after 2 min to see if the gels have developed. Remove the gels just as the bands turn mid-dark purple, usually 2 min for LD, up to 10 min for G6PD, MD and NP. (N.B. Do not let the gels over-develop).

Immediately submerge gels in RO water and leave until background stain disappears (1 to 2 hr).

Dry the gels overnight at 37 ± 2°C.

Fix gels in gel documentation form in the 'Isoenzyme Analysis' file, aligning the origins.

Measure migration distances from the origin in mm. For G6PD, MD and NP calculate ratios using the following equation:

Migration of band for test species

Migration of band for standard (L929)

and compare with expected ratios given in the Authentikit handbook.

For LD, observe the pattern of bands with those given in the Authentikit handbook.

Record details of testing on report sheet AHC/1998/3/3.1/2.2 Appendix 1 for authorisation and further action if necessary.

REAGENTS

Extraction Buffer: 50mM TRIS

1mM EDTA

pH to 7.5 using conc. HCL

Add Triton x 100 to 2% (v/v).

Store at 2 to 8°C for up to 6 months.

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