The importance of antibodies as tools in scientific research studies cannot be understated, yet these reagents have increasingly come under fire for their lack of reproducibility. Part of the issue is that the antibody market is composed of hundreds of vendors and resellers with varying definitions for validation and consistency. Cell Signaling Technology (CST) believes that antibody suppliers should be held accountable for the products they provide, but that vendors alone cannot solve the reproducibility “crisis." How antibodies are validated and used in the laboratory is a critical component to this process. Researchers need to be more attentive to following established protocols and leverage the expertise of the scientists who have developed and tested the product they are using. Journals need to be more active in enforcing existing policies regarding materials and methods or develop more clear-cut means to identify and describe the use of biological reagents in published research. During this webinar we will address the role vendors, researchers, and journals should play in minimizing irreproducibility. We will also outline CST’s antibody validation process, while highlighting steps all users should consider when selecting and using antibodies in their research.
You’re gathering data from all your experiments and preparing to present to your advisor and thesis committee at your annual progress report. You have an interesting hypothesis, and you have a validated antibody that recognizes your target protein on a western blot (WB). The molecular weight of the band is correct, and the expression of the target protein changes just the way you predicted it would. Now, you know — and you’d bet the house on it — when that powerpoint slide comes up, someone on your committee is going to ask about loading controls.
The use of multiple antibodies in a single experiment can provide useful information to researchers. Co-staining with multiple antibodies and cellular dyes is a simple, low-content form of multiplex analysis. Techniques for performing multiplex analyses in cells and tissues are powerful research tools that are applicable to general cell biology studies as well as diagnostic purposes. These techniques allow researchers to detect multiple biomarkers to assess their samples. They also allow for easy colocalization studies to determine relationships between analytes. Here we describe two common techniques for fluorescent staining using multiple antibodies in the same assay.
A picture is worth a thousand words, or in the case of immunofluorescent imaging, a thousand proteins. The images used to illustrate a scientific experiment should convey as much information as the text itself. Here at CST, we pride ourselves in the quality of our antibodies and our rigorous validation process. When we approve our primary antibodies for IF, we like to showcase them using high quality images generated in-house. Beyond our recommended IF protocols (check it out here), here are some additional considerations to make when planning your IF staining.
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.