If you’ve ever transitioned your IHC experiments from a manual protocol to an automated platform, you may have found the conversion process to be a drag. It isn’t an easy thing to do. For that reason, we’re happy to announce our IHC Leadership in Automation initiative. This rigorous validation initiative expands on our already thorough measures, allowing researchers to not only use CST products with our recommended manual IHC protocol, but also to bridge the assay to new platforms and techniques. Our foray into the world of automated IHC aims to reduce the amount of time researchers spend on assay transfer and protocol optimization.
Early exploration of unmapped biological signaling pathways were carried out using radiolabeled phospho-imaging. The development of phospho-specific antibodies to detect and quantify protein phosphorylation made life easier for researchers (less 32P waste to deal with), but the interpretation of data from these experiments comes with its own set of caveats.
well-validated antibody, the workhorse of immunofluorescence. If you are a seasoned pro at IF experiments, you are probably used to checking the antibody datasheet (or web page) for the recommended dilution. But have you ever wondered where those recommendations come from?
The performance of an antibody is a crucial determinant in getting reliable immunofluorescence (IF) results. Equally important is the preparation of the biological sample - cells or tissue used in your experiments - before any antibodies are introduced. The fixation and permeabilization of your samples are key steps that can determine your experiment’s failure or success. The ideal fixative preserves a “life-like” snapshot while quickly stopping the degradative process of autolysis by crosslinking and inhibiting endogenous enzymes. This post provides examples of how different antibodies perform at their best using different protocols.
After months of hard work, your research has honed in on a hypothesis you can test with immunofluorescence (IF). You've chosen antibodies and performed pilot IF experiments (see The Importance of Validation), and the localization of the protein appears reasonable. But how can you be sure the IF data you've acquired represents real biological phenomena? We present two examples of experimental controls in this post.
After months of hard work, your research has zeroed in on a hypothesis you can test with immunofluorescence (IF). But now you have to make a choice. How do you decide which antibody to use to get reliable IF results? How do you know if the images are accurately reporting the target's localization? We explore some considerations in this post.
Antibodies targeted to histone modifications may bind non-specifically to similar, but off-target histone modifications. Conversely, their specific binding can be inhibited by steric hindrance from modifications on neighboring residues. Assays like ELISA, western blot, ChIP, and IF are commonly used to demonstrate antibody specificity and sensitivity, but they cannot clearly predict how an antibody will interact with nearby epitopes. As a result, an alternative approach is needed when trying to validate an antibody to a histone modification target. Continue reading to see how we do it.
If you buy primary antibodies from different places, you may have noticed that many companies list the size of their products in micrograms per ml (i.e., by weight). You may have also noticed that CST does not - CST antibodies are listed by performance (e.g., 10 western blots) instead.
Thinking about lab expenses isn’t as enjoyable as investigating your favorite signaling pathway. However, because research money is hard to come by, it is something that should be considered when picking your reagents for western blotting. It makes sense to keep the quality of your primary antibody in mind, because the success of the entire experiment depends on the antibody being reliable, specific, and sensitive. If the antibody does not perform as expected your experiment may fail . . . and the cost of a failed western blot may be more than you think.
Antibody Validation Roundtable: Specificity + Sensitivity + Reproducibility = Validation
Concerns about the use of incorrectly or insufficiently characterized antibodies in biomedical research have been raised by recent reports of nonreproducible research studies. Non-reproducible research studies have resulted in loss of time, resources, and scientific reputation, with publication retractions occurring in certain cases.