The Choice of Secondary Antibody is one of the Most Overlooked Issues in Immunochemistry. After the tissue of interest has been incubated with the primary antibody, the next critical step involves incubating the tissue in a labeled secondary antibody to detect the presence of the primary antibody. The choice of the secondary antibody has to […]
As we have discussed previously in this blog, there is widespread concern about antibody validation. Of course some forms of validation using multiple antibodies or knockouts are not available for the majority of targets of interest to researchers. But western blots are a excellent form of validation that can be readily performed by almost […]
In the past year there have been a number of articles in Nature and other major journals that discuss the antibody problem in somewhat apocalyptic terms. In my mind there are only two main issues in the antibody problem: antibody validation and antibody variability. Both of these issues have straightforward solutions that do not […]
Antibody validation is a topic that has garnered a great deal of attention lately in discussions of the problem with the lack of reproducibility in science. One type of antibody validation that should be avoided uses purified recombinant proteins. In a typical study of this type a purified recombinant protein is run on a western […]
In a recent article in Nature Methods, Vivien Marx discussed some of the key issues relating to the use of antibodies http://tinyurl.com/osygswn. Variability in the quality of the antibodies available commercially was one of the principal problems encountered by scientists in her article. This problem is closely related to the larger issue addressed in […]
What does it mean when an antibody company says an antibody “does not work” in western blots? I started wondering about this question the other day after a colleague told me about a company that “validates” all of their antibodies by immunostaining in no less than 5 different tissues! We examined the company’s […]
It is common to see references to the “specificity” or “greater specificity” of monoclonal antibodies compared to polyclonal antibodies. Is that claim justified? No one would dispute the fact that a monoclonal is directed toward a single site or epitope of the target protein, while a polyclonal may contain antibodies directed toward multiple epitopes on […]
Antibody specificity: The use of a blocking control has only limited value Antibody specificity is one of the key issues in determining whether you have an antibody that works. How does one determine that the antibody specifically recognizes only the target of interest? There are a number of control procedures one can use to be […]
1% SDS is the lysis buffer of choice for most western blots or the case of the missing protein in western blots. As mentioned in my opening blog, good antibodies sometimes do not work because of poor technique. One of the most common problems of this type is the failure to solubilize cellular proteins in […]
Antibodies that work are extremely valuable research tools. However, the search for such antibodies is made quite difficult by two different problems. The first problem, “the good antibody problem” is that production of a good antibody is a difficult, time consuming process; and many researchers and antibody companies do not possess either the patience […]