Lab Expectations

 Part two of a four-part series on Immunofluorescence. Check out our posts on Validation and Fixation and Permeabilization. 

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.

Part one of a four-part series on Immunofluorescence. Check out Experimental Controls and Fixation and Permeabilzation.

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.

Specificity, consistency, and optimized assay conditions are three key elements that help ensure reliable immunofluorescence (IF) staining results each and every time. So, before starting an experiment you should ask yourself the following three questions:

• Is your antibody specific?
• Is your antibody supported by an optimized IF protocol?
• Is your antibody performing consistently?

Let's take them each in turn...

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.

Specificity, consistency, and optimized assay conditions are three key elements that help ensure reliable immunofluorescence (IF) staining results each and every time. So, before starting an experiment you should ask yourself the following three questions:

• Is your antibody specific?
• Is your antibody supported by an optimized IF protocol?
• Is your antibody performing consistently?

Let's take them each in turn...

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.

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. 

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.