Isozyme analysis

Summary

Cells were collected, washed with D-PBSA and reconstituted into a cell suspension of 5×107 cells/ml, cell extracts were prepared and stored at -70℃. The gel device was prepared and 1~2 μl of the standard and control spot samples were taken on an agarose gel. Prepare the gel device, take 1~2 μl of cell extract, standard and control spot samples in agarose gel. Fill the tank with liquid, place the agarose gel in it, electrophoresis for 25 min, add the enzyme reaction reagent, incubate for 5-20 min, rinse the gel, dry it, and check the display of the enzyme zone on the completed gel.

Operation method

Program 16.10 Isozyme Analysis

Principle

Cells were collected, washed with D-PBSA and reconstituted into a cell suspension of 5×107 cells/ml, cell extracts were prepared and stored at -70°C. The gel device was prepared and 1~2 μl of the standard and control spot samples were taken on an agarose gel. Prepare the gel device, take 1~2 μl of cell extract, standard and control spot samples in agarose gel. Fill the tank with liquid, place the agarose gel in it, electrophoresis for 25 min, add the enzyme reaction reagent, incubate for 5-20 min, rinse the gel, dry it, and check the display of the enzyme zone on the completed gel.

Materials and Instruments

Cells Extracts Enzyme substrates Nucleotide phosphorylase (NP) Glucose 6-phosphate dehydrogenase (G6PD) Malate dehydrogenase (MD) Lactate dehydrogenase (LD) Aspartate aminotransferase (AST) Mannose 6-phosphate isomerase (MPI) Peptidase B (Pep B)
Cell extraction buffer or Triton X-100 extraction solution Buffer SAB8.6 D-PBSA Distilled water Deionized water
Authentikit devices and reagents Agarose gel membranes SAB8.6 Incubation tray liners Incubation trays Staining or washing trays Tank lids and bottoms of temperature-controlled chambers Timing electrophoresis apparatus Safety connectors Incubation chambers or desiccators Magnetic stirrers with 1-inch-long magnetic rods Sample spikers Teflon heads Gel log sheets Enzyme migration data sheets Cell I.D. final analysis sheets Microliter syringes Microcentrifuges Eppendorf tubes Sampler Sampler tips Pipettes 100 ml graduated cylinder Marker pens Protective gloves Trash can for tips

Move

I. Preparation of extracts

1. Cultivate the cells up to ^2×107 viable cells according to the conventional method.

2. Collect the cells according to the recommended methods for specific cell line experiments.

3. Resuspend the cell pellet in D-PBSA and count the viable cells.

4. The cell suspension is centrifuged at 300 g for 5-10 min to precipitate the cells and the supernatant is decanted.

5. Repeat steps 3 and 4 for a total of 3 washes.

6. Add 100 μl of extraction solution (cell extraction buffer (Innovative Chemistry, Inc.) or Triton X-100 extraction solution: 1:15 (V/V), Triton X-100 in saline, containing 6. ^6×10-4 mol/L EDTA, pH 7.1 (stored at 4°C)) to the cell pellet.

7. Carefully aspirate the cell suspension into a small-volume pipette and blow up and down until all cells are lysed.

8. Transfer the cell lysate to an Eppendorf tube (1.5 ml), blow up and down with the pipette for several minutes until the cell membranes become cloudy and clustered together, centrifuge the cell lysate with a microfuge at the highest speed (about 9000 g) for 2 min, collect the supernatant, aliquot into the desired volume, and store it at -70 °C.

Installation of the electrophoresis unit

1. Turn on the incubator.

2. Place a tray mat in each incubation tray.

3. Add 6.0 ml of deionized water to each incubator tray and hold at 37°C for 20 min.

4. Add 95 ml of SAB 8.6 buffer to each tray chamber of the electrophoresis tank (total volume of buffer in the entire tank is 190 ml). This buffer is not reusable because the pH value changes significantly during electrophoresis.

5. Connect the electrophoresis tank with buffer to a power supply with adjustable voltage via the safety cable and ground the power supply.

6. Fill the surface of each temperature-controlled electrophoresis chamber cover with 500 ml of cold water (4~10°C). The electrophoresis lids must be filled with water when placed vertically, otherwise the water will be lost. Cool the water with ice or store enough cold water in the refrigerator and replace the cold water before each electrophoresis.

7. Add 500 ml of deionized or distilled water to each staining tray and wash the incubation tray with it. This water cannot be reused.

Steps of electrophoresis

1. Label each agarose gel to be used. Mark the back of the gel with Innovative Chemistry, Inc. labels or with a permanent marker.

2. Place the gel on the bench with the plastic protrusions down. Orient the gel so that the sample wells are closest to the operator.

3. Label each sample well with the sample to be tested, (e.g., Standard, Control, Unknown 1, Unknown 2, etc.) 6 unknown samples can be tested per gel.

4. Carefully peel the agarose gel away from the hard plastic support. Handle the gel carefully along the edges and discard the hard plastic support.

5. Add cell extract to the sample wells. Precisely add 1 μl of Cell Extract to each well using a dispenser fitted with a Teflon tip. Use a new head for each sample. Dispense molecular weight standards into lane 1, controls into lane 2, and unknowns into lanes 3 to 8. To avoid damaging the agarose, only the droplet of cellular extract should touch the wells; the tip of the dispenser should not touch the wells. To avoid damage to the agarose, only the drop of cell extract should touch the sample wells, and the tip of the dispenser should not touch the wells. If the sample volume is 2 μl, the second drop should be dispensed after the first drop has been dispersed into the agarose.

6. Insert each dispensed agarose gel into the lid of the electrophoresis tank with the agarose side out. Match the anode (+ ) of the agarose gel to the anode (+ ) of the electrophoresis tank lid. It may be necessary to bend the agarose gel slightly in order to insert it into the electrophoresis tank lid.

7. Place an electrophoresis tank cover on each electrophoresis tank holder. The black end with the magnet inside is closest to the power supply. Turn on the power (160V) and time it for 25 min.

8. About 5 min before the end of the electrophoresis, take out each substrate reagent bottle from the refrigerator and bring it back to room temperature.

9. Add 0.5~1 ml of deionized water to each vial before use, and gently swirl the vial to dissolve the reagent.

10. At the end of electrophoresis, remove the lid from the electrophoresis tank holder and place it on absorbent paper.

IV. Staining Steps

1. Remove the agarose gel from the electrophoresis tank chamber by grasping the edge of the gel membrane and gently pressing it inward to remove it from the lid.

2. Place the agarose gel on its side on absorbent paper placed horizontally on a flat surface with the spotting hole facing the operator.

3. Carefully remove any residual buffer from the ends of the agarose gel with lint-free blotting paper.

4. Place a 5 ml pipette along the bottom edge of the agarose gel membrane.

5. Pour the reconstituted substrate evenly onto the agarose gel along the leading edge of the pipette.

6. Push the pipette past the agarose surface in a smooth motion, one at a time, and then drag the pipette back toward the operator, pushing several times on the agarose surface as the pipette rolls away from the end of the agarose, removing excess material in the process, being careful not to damage the agarose gel; no pressure is required for this step.

7. Place the neatly edged agarose gel in a preheated incubator dish with the agarose side up and place the dish in an incubator at 37 °C for 5-20 min.

8. After incubation, wash the agarose gel twice with 500 ml of distilled or deionized water, stirring with a magnetic stirring bar for 15 min each time, and after the first 15 min, remove each gel film, discard the water, and add 500 ml of fresh water to a vessel, submerge the gel film in the water, and cover from the light to ensure that the gel film is fully immersed and not on the floating wash solution.

9. Remove the gel film from the water and place it on a drying rack in an incubator or oven drying chamber and dry for 30 min or until the agarose gel is dry, or the agarose gel may be dried overnight at room temperature.

10. Clean and organize, pour off the bottom buffer of the electrophoresis tank and rinse with distilled water, pour out the water from the electrophoresis lid, rinse the inside of the lid, and air dry.

11. Evaluation of results, zones are permanent and gel membranes can be saved for future reference, if background staining occurs over time, it indicates insufficient gel cleaning.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/