Antibody Specificity Validation

In the field of life science research, antibodies play a crucial role as powerful tools for detecting, isolating, and visualizing specific antigens. However, the reliability and accuracy of antibodies have been a subject of concern in recent years, and the cross-reactivity and variability of antibodies have led to the "reproducibility crisis" or "antibody crisis". To address these issues, Creative Diagnostics is committed to providing highly validated antibodies that ensure specificity and accuracy in various applications.

Browse all Primary Antibodies

Browse all Secondary Antibodies

Browse all Control Antibodies

Validation Methods for Antibody Specificity

Creative Diagnostics employs a rigorous validation process to ensure the specificity of our antibodies. Here, we discuss several key methods used for antibody specificity validation:

• Knock-Out (KO) Validation
  The gold standard for antibody specificity validation is the use of KO cells or tissues. By comparing the signal obtained from wild-type samples to KO samples, the absence of signal in the KO sample confirms the specificity of the antibody for its intended target. However, it is important to note that KO validation in western blotting does not guarantee specificity in other assays like immunostaining. Unfortunately, KO cells or tissues are not always available.
  Browse all knockout-validated antibodies
• Knock-Down (KD) Validation
  KD experiments, achieved through RNA interference (RNAi), can also provide valuable insights into antibody specificity. Reduction in signal intensity in KD samples compared to control samples indicates the antibody's specificity for the target protein. However, KD is rarely 100% effective, thus good controls and careful interpretation of results are necessary.
• Blocking/Pre-Adsorption
  In the case of polyclonal antibodies, blocking or pre-adsorption experiments can be performed. This involves incubating the antibody with the immunogen used for immunization. If the signal disappears after pre-adsorption, it suggests that the observed signal is specific to the target antigen. However, the possibility of cross-reactivity with other proteins sharing similar binding epitopes cannot be completely excluded.
• Transfection of Cells for Target Overexpression
  By overexpressing the target protein in cells that exhibit weak or no endogenous expression, the specificity and sensitivity of an antibody can be assessed. Detection of the target protein at the expected size or localization in combination with a positive result in the overexpression system provides evidence of antibody specificity.
• IP/MS Validation
  Immunoprecipitation (IP) followed by mass spectrometry (MS) involves first immunoprecipitating protein complexes from cell lysates and then analyzing the protein complexes by MS. IP-MS is a powerful tool for analyzing antibody specificity for IP or ELISA applications. It identifies all protein isoforms bound by the antibody and confirms specificity based on digested protein fragments. However, this approach does not yield much information about antibody specificity in other applications such as immunostaining or Western blotting.
• Western Blot
  The method involves detecting proteins in a sample through initial size separation, followed by blotting to a membrane for visualization with antibodies. Western blotting determines antibody specificity for a target protein based on molecular weight and is ideal for detecting reduced/denatured proteins. However, this method is time-consuming and only detects a small number of antibodies per experiment.
• Immunocytochemistry and Immunohistochemistry
  In both methods, proteins in cells or tissues are detected using specific antibodies and reporter molecules. Specificity depends on whether the cells or tissues express the protein of interest. However, these two methods cannot determine whether the antibody recognizes other proteins with the same cellular localization.
• Protein/Peptide Array
  The method involves detecting antibody binding events by first spotting the array with peptides/proteins and then adding antibodies. This method can screen a large number of overexpressed proteins, but cannot screen post-translationally modified proteins.
  In the realm of life science research, antibody specificity validation is of paramount importance to obtain accurate and reliable results. To meet this objective, Creative Diagnostics implements a comprehensive validation process that encompasses multiple validation methods tailored to different applications. By extensively characterizing and validating our antibody products, our researchers aim to provide researchers with antibodies of the highest quality and reliability. By choosing Creative Diagnostics as your antibody supplier, researchers can confidently rely on the quality and reliability of these essential tools in their scientific endeavors.