What is Cellulose Acetate Electrophoresis?
Cellulose Acetate Electrophoresis (CAE) is a method used for separating and analyzing proteins based on their charge and size. It involves the migration of proteins through a cellulose acetate membrane under the influence of an electrical field.
The significance of CAE lies in its ability to separate proteins with a high degree of resolution, allowing for the detection of minor differences in protein composition. It is a relatively simple and inexpensive technique, making it accessible to a wide range of researchers.
The applications of CAE are diverse and include:
- Clinical diagnosis of protein abnormalities in serum and other biological fluids
- Characterization of protein isoforms and variants
- Analysis of protein-protein interactions
- Purification and identification of proteins for structural and functional studies
- Detection and quantification of protein contaminants in food and pharmaceuticals.
Cellulose Acetate Electrophoresis Protocol
1. Starting Material: The starting material can be a sporulating lesion or a sporulating Phytophthora infestans culture.
A. For sporulating lesion, place the lesion in an Eppendorf tube with 1 ml of dH20, vortex the sample to release the sporangia, and spin the tube on high for 3 minutes. Keep one drop of liquid and proceed to step 2.
B. For sporulating culture, place a small amount of sporulating mycelium in an Eppendorf tube containing 2-3 drops of dH20. Proceed to step 2.
2. Homogenization: Grind the samples for 30-60 seconds using a homogenizer attached to an electric drill to break apart the sporangia. Spray the homogenizer with dH20 and wipe down with a Kimwipe between samples.
3. Loading Samples: Pipette 8-10 ul of each sample in a different well of the sample well plate. Note which sample was placed in each well on a separate sheet of paper.
4. Blotting Plate: Blot a cellulose acetate plate between two pieces of Whatman paper soaked in buffer solution. Align it on the aligning base with cellulose acetate side up.
5. Applying Samples: Align the applicator with the sample well plate and press down several times to pick up some of each sample on the applicator. Then align the applicator on the aligning base and press down 2-3 times to transfer the material from the applicator to the plate. Repeat this entire process 2-3 times.
6. Adding Dye: Place a small amount of a running dye, usually food coloring, to spot one side of the gel to monitor the electrophoresis.
7. Electrophoresis: Place the plate cellulose acetate side down in the gel box containing buffer solution so that the plate is in good contact with the 2 wicks and the wells are closest to the – side of the chamber. Run the electrophoresis for 18-20 minutes at 200 V.
8. Preparation for Staining: Five minutes before the end of electrophoresis, prepare the following solution:
A. Make a 1.6% agar solution (160 mg/ml).
B. Combine the following for each gel. Wear gloves!!!
- Tris-HCl, 0.05 M, pH 8.0 1.5 ml
- Fructose-6-phosphate, 20 mg/ml 5 drops
- NAD, 3 mg/ml 1.0 ml
- MTT, 10 mg/ml 5 drops
- PMS, 2 mg/ml 5 drops
9. Staining: After electrophoresis is over, place the plate face-up on a glass plate. Add the solution made in step 8B to it. Agar, 1.6%, 60oC, and G-6-PDH, 1 U/ul, 3.0 ul. Pour this solution over the plate and allow the reaction to take place. Bands will become visible on the gel. Once the bands have fully developed, wash the solution off using dH20, and the plate may be photographed.