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Many customers have reported that what seems like a simple ELISA experiment can be somewhat challenging in practice, leading to various issues that affect detection accuracy and test quality. Problems such as background noise, false positives, non-specific binding, and low signal-to-noise ratios are common. These challenges highlight the importance of carefully following experimental procedures, continuously refining techniques, and focusing on key steps in the ELISA workflow.
First: Selection of Coating Solution
The most commonly used coating buffer is carbonate buffer at pH 9.6. However, depending on specific testing requirements, other buffers such as phosphate buffer or Tris-HCl (pH 7.2–7.4) may also be suitable. When choosing a coating solution, it's important to consider the nature of the antigen or antibody being immobilized. Proteins typically bind to polystyrene solid-phase carriers through physical adsorption, which is influenced by hydrophobic interactions between the protein and the surface. Larger molecular weight proteins with more hydrophobic regions tend to bind more strongly. Understanding these principles helps optimize the coating process for better results.
Second: Blocking
After coating, blocking is an essential step to prevent non-specific binding. This involves filling the remaining active sites on the plate with a high concentration of unrelated proteins. Common blocking agents include 0.05%–0.5% bovine serum albumin (BSA), 10% or 1% gelatin, and skim milk powder (which is cost-effective and suitable for higher concentrations, such as 5%–10%). In some cases, animal sera may be used to minimize cross-reactivity. The choice of blocking agent should be based on the specific needs of the experiment and the type of detection system being used.
Third: Washing the Plate
Washing is one of the most critical steps in ELISA. Due to the tendency of proteins to adsorb onto plastic surfaces, thorough washing is necessary to remove unbound enzymes, free antigens, and non-specifically bound substances. Poor washing can lead to high background signals and reduced sensitivity. Manual washing is prone to human error, such as incomplete rinsing or over-washing, which can significantly impact the results. It’s recommended to use automated washers when possible for consistent and reliable performance.
Fourth: Adding Antibodies (Primary and Secondary)
When adding antibodies, care must be taken to avoid air bubbles and ensure accurate pipetting. Both primary and secondary antibodies should be diluted appropriately using the specified diluent. If working with two antibodies, it's crucial to determine the correct working concentrations to avoid excessive background or weak signal development. Proper dilution ensures optimal binding and enhances the overall reliability of the assay.
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