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Cat. No.: RWGAP-15 (for 6-15T)

Cat. No.: RWGAP-75 (for 30-75T)

Description

Rapid Western Assay Kit for GAPDH is an ultra-fast Western blot detection kit that allows for the visualization of the internal reference protein, GADH, in just 10 minutes from a completed transfer blot. This kit includes Anti-GAPDH-HRP, Blocking and Antibody Dilution Buffer, Western Washing Buffer, and the ECL Star Chemiluminescent Reagent Kit. The primary and secondary antibody incubation steps are combined into a single step, making it convenient to use with low background noise. It contains all the necessary reagents from blocking to chemiluminescence, enabling high-quality internal reference protein Western results typically within 10 minutes after membrane transfer, significantly speeding up the detection process.

The kit is compatible with the detection of GAPDH from humans, mice, and rats.

Internal reference proteins are constitutively expressed in various tissues and cells, with relatively constant expression levels that are minimally affected by external inducers. Therefore, they are widely used as a loading control to ensure consistent protein sample loading in Western blots. However, different cells, tissues, conditions, and even varying protein sample amounts can impact the detection of internal reference proteins, so it is recommended to test one or more internal reference proteins.

This series of products currently includes the Actin/GAPDH/Tubulin Superquick Western Assay Kits.

Blocking and Antibody Dilution Buffer included in this kit can be used for both membrane blocking and antibody dilution. Blocking time is only 1 minute, and the overall effect is significantly better than traditional blocking buffers based on BSA (bovine serum albumin), nonfat milk, or casein. The working solution of the internal reference antibody prepared and diluted using this product can be stored and used at 4ºC for at least one month and can be reused.

This kit uses Anti-GAPDH-HRP, allowing for the primary and secondary antibody incubation to be completed in a single step. The antibodies have high specificity and excellent sensitivity, with optimized usage concentrations. Diluted at a 1:500 ratio, they typically require a 5-minute incubation at room temperature before subsequent washing and detection.

ECL Star Chemiluminescent Reagent provided in this kit offers high sensitivity, low background, and long-lasting luminescence, suitable for most routine Western detections. In special cases where imaging optimization is needed, higher sensitivity ECL Moon or ECL SuperMoon can be selected based on current experimental results.

Using this kit, the actual operation from a completed transfer blot takes about 10 minutes.

Components

• Anti-GAPDH-HRP
• Blocking and Antibody Dilution Buffer
• Western Washing Buffer
• ECL Star Solution A
• ECL Star Solution B

Specification

If detecting a 6.6×8.5cm blot membrane, each Western blocking requires 5ml of Blocking and Antibody Dilution Buffer, and antibody dilution requires 5ml of Blocking and Antibody Dilution Buffer plus 10μl of antibody. The diluted antibody can be used a total of three times. Washing buffer is used at 10ml per wash, and the chemiluminescent reagent working solution requires 1ml per use. Based on this, the small and medium kit packages can perform 6 and 30 GAPDH Western rapid detections, respectively.

For the more common cut blot membranes of approximately 2×8.5cm, each blocking and antibody incubation requires 2ml of solution, 4ml of washing buffer per wash, and 0.4ml of chemiluminescent reagent working solution per use. The small and medium kit packages can perform 15 and 75 detections, respectively.

Storage Conditions

Store at -20ºC for one year. Blocking and Antibody Dilution Buffer, Western Washing Buffer, and ECL Star Solution A and B should be stored at 4ºC and are valid for at least six months.

Notes

• This product is suitable for detecting GAPDH in humans, mice, and rats. For other species, a preliminary experiment is recommended.
• Please recycle the used antibody working solution. The recycled antibody working solution can be reused up to two more times. Store diluted antibodies, including used working solutions, at 4ºC.
• Recycled antibody working solutions should be used in the same way as freshly diluted antibodies. If slight turbidity is observed during reuse, centrifuge at 10,000×g for 1-3 minutes and use the supernatant for subsequent detection. If the recycled antibody working solution shows significant flocculation or mold/bacterial growth, consider discarding it.
• Using this product, high-quality internal reference protein Western results can typically be obtained within 10 minutes, which includes 1 minute for blocking, 5 minutes for antibody incubation, three 30-second washes, 1 minute for ECL reagent incubation, and imaging usually within 0.5 minutes.
• For 6.6×8.5cm blot membranes, it is recommended to use Western Washing Box for washing and incubation; for cut strip blots, 5-slot Western Washing Box is recommended.
• In some cases, to achieve better Western results, you may increase the time for blocking, antibody incubation, and washing, and optimize the exposure time for imaging as needed.
• Both ECL Star Solution A and B are harmful to humans. Handle with care and ensure effective protection to avoid direct contact with skin or inhalation.
• This product is for scientific research use by professionals only. It is not intended for clinical diagnosis or treatment, food, or drug use, and should not be stored in a residential setting.
• For your safety and health, please wear a lab coat and disposable gloves during operation.