Preparation of Reagents and Solutions
0.1mol/L Barium Acetate Buffer (pH 5.0)
- Take approximately 25.24g of barium acetate.
- Dissolve it in water and dilute to 900ml.
- Adjust the pH to 5.0 using acetic acid.
- Add water to make the total volume up to 1L and mix well.
Staining Solution: 0.1% Toluidine Blue Acetate Solution
Take 1g of toluidine blue.Dissolve it in 0.1mol/L acetic acid solution to a final volume of 1000ml.
Standard Solution 1
Prepare a 30mg/ml solution of sodium chondroitin sulfate reference standard in water.
Standard Solution 2
Take 1ml of Standard Solution 1.
Dilute it with water to 50ml and mix well.
Test Solution
- Weigh 150mg of the test sample accurately.
- Dissolve it in water and transfer to a 5ml volumetric flask.
- Dilute to the mark with water and mix well.
Electrophoresis Procedure
Electrophoresis Cell Setup
- Use an electrophoresis unit suitable for Cellulose Acetate Membranes.
- Fill the electrophoresis cell with 0.1mol/L barium acetate buffer (pH 5.0).
Preparation of Cellulose Acetate Membranes
- The size of Cellulose Acetate Membranes is approximately 5×14cm or 6× 12cm.
- Immerse the membrane in the barium acetate buffer (pH 5.0) for 10 minutes or until completely soaked.
- Remove the membrane and blot it with filter paper to remove excess buffer.
Loading Sample
- Place the membrane on the electrophoresis rack with the smooth side facing up.
- Using a suitable samples loading device, apply 0.5μl of the test solution, Standard Solution 1, and Standard Solution 2 onto the membrane.
- Ensure that at least 0.5~1.0cm of the membrane at each end is immersed in the buffer solution.
Cellulose Acetate Membrane and sample application device
offered by Beijing Liuyi Biotechnology
Electrophoresis Process
Perform electrophoresis at a constant voltage of 60V for 2 hours, with an initial current of approximately 6mA.
Note: The application of samples must be completed within 5 minutes, and the electrophoresis should start immediately to prevent drying of the membrane, which could reduce its sensitivity.
Staining and Result Analysis
Staining and Washing
- After electrophoresis, place the membrane with the applied side facing down.
- Gently agitate or immerse the membrane in the staining solution for 5 minutes.
- Stir the solution gently for 1 minute to ensure even staining.
- After staining, remove the membrane and rinse it with 5% acetic acid solution until the background color is completely removed.
Comparison and Analysis of Results
Comparison of Color Bands
The main color band of the test sample should be in the same position as the main color band of Standard Solution 1.
The color band of Standard Solution 2 should be clearly visible, and its migration rate should match that of Standard Solution 1.
Secondary Color Bands
All secondary color bands in the test sample should not be darker than those in Standard Solution 2.
Detection of Any Impurities
Any single impurity band detected should not exceed 2% in intensity.
Recording Results and Judgment
Take a photograph of the result within 15 minutes after decolorization.
Beijing Liuyi Biotechnology Co., Ltd. (Liuyi Biotechnology) has over 50 years of expertise in manufacturing electrophoresis instruments. With a dedicated technical team and R&D center, we provide reliable and comprehensive production, from design to inspection and warehousing. We offer a full range of products, including:
- Electrophoresis Cells (Tanks/Chambers)
- Electrophoresis Power Supplies
- Blue LED Transilluminators
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Post time: Dec-03-2024