12/31/2023 0 Comments Controls for antibody cross reactivity![]() (B) Lysates prepared from lung tissue or tracheal epithelial cells were immunoblotted to detect β6 by using a panel of antibodies. (A) Mouse lung sections collected at 3 dpi were stained for β6. Specificity of β6 antibodies in immunofluorescent microscopy and western blot analysis. Fortunately, we received a highly specific antibody from a collaborator (control antibody) and stained tissues successfully without detecting signal in sections from KO mice.įig 1. Slides stained with only secondary antibody were negative, suggesting that the signal was not due to nonspecific binding of the secondary antibody. However, we also detected a weaker yet concerning signal in confirmed β6 knockout (KO) mice ( Fig 1A). Initially, we detected a strong signal by IFA. We used antibody 1 for immunofluorescence (IFA) staining of mouse pulmonary tissues to detect β6. In our work, we purchased several commercial antibodies from different companies to evaluate specificity. It is especially difficult to generate antibodies against specific integrins due to their similar structures, and many specification sheets report a small degree (10%–20%) of cross-reactivity with other integrins and proteins. The specificity of antibodies to receptors and proteins implicated in cell signaling is not well defined in the literature. Our laboratory recently published a study focused on β6 integrin (β6), a small heterodimeric molecule involved in cell signaling. In one report, an mAb targeting the Met tyrosine kinase receptor-a marker of breast cancer diagnosis-revealed the target protein in the nucleus, while another lot showed membrane and cytoplasmic staining. Additionally, epitopes that mAbs target are generally short sequences of amino acids that might exist on other proteins. A hybridoma might also lose its antibody gene through continued passaging. Hybridomas maintained in ascites can be contaminated with endogenous immunoglobulins and other proteins, especially if the mAb is not purified. ![]() mAbs, although generally more consistent, are not exempt from variation. However, it is possible to reduce nonspecific binding of a pAb via immunoaffinity enrichment. Each lot, even when prepared from the same donor animal, contains diverse antibody clones and concentrations. pAbs are a heterogeneous mixture of antibodies that recognize multiple epitopes of the same target protein but can also include nonspecific antibodies. ![]() Problems with reproducibility often arise due to lot-to-lot variability and affect both polyclonal (pAbs) and monoclonal antibodies (mAbs). ![]() Sensitivity is especially problematic with low-abundance proteins, for which the antibody in question can only detect high levels of target. To determine whether an antibody is suitable, the following three issues must be considered: ability to detect the target (specificity), detection of the target above background (sensitivity), and generation of consistent results (reproducibility). Multiple studies have focused on issues of antibody specificity towards proteins such as G-protein-coupled receptors, kinase receptors, and integrins. Currently, there is no standard for validation and reference data that must be provided in publications, and crucial specificity data are often unavailable. However, recent concerns have resulted in a National Institutes of Health (NIH) mandate to vigorously test the specificity of antibodies used in publications ( ). Commercial antibodies offer a convenient solution. The process of making antibodies is costly and time-consuming. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |