Western Blot, also known as Western blotting, is an experimental method for the quantitative or qualitative detection of proteins based on specific immune responses between antibody antigens commonly used in molecular biology, biochemistry and immunogenetics.

Basic principle: Polyacrylamide gel electrophoresis separates protein samples of different molecular weights, and the separated proteins are transferred to a solid support (hybrid membrane), and the antibody specifically recognizes the target protein by enzymatic reaction or chemiluminescence. The target protein is visualized to qualitatively or quantitatively study the protein.

The specific recognition between the antibody and the antigen is the most critical step in the Western Blot experiment. Whether the selected antibody is suitable or not, the quality of the antibody is related to the success or failure of the experiment. The following will give some suggestions on the selection of high-quality antibodies, the importance of the verification data for antibodies, and the selection criteria for internal reference antibodies.

√ Quality is the primary criterion for selecting quality antibodies

Commonly used antibodies in the market include imported brands, domestic brands, and imported brands. Most imported brands are more secure in terms of antibody quality, specificity, potency and storage conditions. However, some domestic brands of antibodies, when doing Western Blot, due to their low specificity, resulting in experimental results in the background is not clean, non-specific bands and other issues. The domestic packaging of imported brands is after individual dealers buy the products from the manufacturers, and then sell them to customers after dilution, repackaging and re-branding. Although the prices of such antibodies are relatively cheap, the quality is not uniform, and the batches vary greatly.

To ensure the high quality of antibodies, we usually choose a reliable brand. In addition, we should also pay attention to whether the supplier channel of the antibody is formal and whether the supply chain logistics is professional.

完善 Perfect verification data is a necessary reference for antibody selection

High-quality antibodies should also have comprehensive validation data and detailed information annotations, such as whether it is clearly labeled for Western Blot, whether there is sufficient data to support the application of the antibody to a tissue or sample, and whether there are clear antibody recommendations. The dilution concentration, the recognition site of the antibody is the primary structure of the continuous amino acid sequence or the three-dimensional structural site of the discontinuous amino acid... The authenticity and integrity of the information and data are the key to the smooth progress of the Western Blot experiment.

In this regard, HPA gold-labeled antibodies and the data they provide are a good example. The HPA antibody database covers 85% of human proteins, and each antibody passes through stringent antigen sequence screening and affinity chromatography purification procedures to ensure its specificity and purity. In addition, each antibody was tested in a variety of tissue samples by protein chip, Western Blot, immunohistochemistry, immunofluorescence and other experiments, and obtained the certification of the academic committee. As an important reference data, all experimental validation, published literature and bioinformatics predictions are published on the proteinatlas.org website for free reference. Complete experimental data support avoids selection errors caused by incomplete information.

√ Effective internal reference antibody is an important and accurate testimony

In Western Blot, the internal reference is a housekeeping proteins encoded by the housekeeping gene. The expression in the tissues and cells is relatively constant, and is often used as a reference when detecting changes in the expression level of the protein. The use of internal parameters to correct protein quantification and experimental errors in the loading process is an important evidence to prove that the results of Western Blot experiments are true and reliable. Some common internal reference genes and antibodies include β-actin, GAPDH , PCNA , α-Tubulin and the like.

It is worth noting that different internal reference antibodies are only suitable for a range of samples and tissues. We need to consult the reference and select the appropriate internal reference antibody according to the difference between the sample and the tissue to ensure the accuracy of the experiment. (as shown in the table below)

In Western Blot, if the antibody is selected or used improperly, it may cause experimental problems such as no signal, weak signal, high background, and miscellaneous band. The following table gives case analysis and solutions for such experimental problems for reference.

These are some suggestions on how to choose the right, high-quality antibodies in the Western Blot experiment, as well as a summary of the problems caused by the lack of antibody selection and use, I hope to give you some help and inspiration.

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