Author: gebauer

Plate layout & proper controls

This entry is part 1 of 1 in the series ELISA

In scientific experiments in general and in ELISA-style binding assay in particular, proper controls are of utmost importance. Without these an interpretation of the final result is often impossible. In planning a proper plate layout, you ensure that the signals you see, are really caused by your protein interaction and not by non-specific binding (NSB is a general problem in many interaction-assay we will later spend and whole article on.)

To detect non-specific binding, you need at least these controls: read more

How does an ELISA work and which protein to coat?

In this article, I shortly explain the basic principle of an ELISA style binding assay and discuss the first considerations in creating an ELISA style binding assay.

How does an ELISA work?
Normally assays are executed in 96 well plates (very common: NUNC Maxisorp). One protein is immobilised to the plastic by hydrophobic interactions and the remaining bindings sites are blocked by a blocking agent. Subsequently the second protein is exposed to the well either in a concentration series for determination of affinity constants or as a single concentration for screening purposes. After washing away unbound proteins, the bound protein is detected specifically by an antibody (in our days often tag specific). This first antibody is then detected by a secondary antibody coupled to an HRP or a fluorescent dye, which in turn either catalyse a colour reaction or is directly measured by a fluorescent reader.
Initial thoughts…
To develop an ELISA assay, you first decide on which protein will be coated (the ligand) and which will stay in solution. Ideally, you try both options – but this is often not possible:
1) The coated protein (=ligand)
The ligand (the protein you are going to coat) should be rather pure. If you use an unsual ligands (like DNA, peptides or membrane proteins) you need to check, whether your compound really immobilises on the ELISA plate (oligos and small peptides do normally not!). If not use you might try a different plastic surface or perform a sandwich ELISA [link]. In my experience apart from the above mentioned exceptions coating is not problematic – however, if it is (anticipated), the best way around the problem is to use the protein as the analyte instead
2) The soluble protein (=analyte)
The analyte does not need to be as pure as your ligand, as you will detect it specifically with the antibody. It is not unusual to use blood serum or other complex mixtures as the analyte. However, as you normally have no way to know the proper concentration of your protein in complex mixtures, only simple yes/no answer can be derived from this kind of assay (1). To detect your bound protein, you need a specific antibody. If you use a protein tag as an epitope, you have to be sure that your ligand does not have the same epitope. Cutting protein tags off, might sometimes alleviate this problem; however, in my experience there is often enough uncut protein left to give strong “background” problems. If you have no good antibody, biotinylation [link] of your protein might help, although this procedure might turn your protein incapable of binding. Your ligand should not tend to aggregate or precipitate, as this will give false positive signals. You might consider using such a protein (like collagens, which I use a lot) as coat instead.
Additionally, protein amounts might also influence your decision. While you use only 500ng of your protein per well for coating typically 2-4 µg are necessary for high analyte concentration. read more

ELISA-style-binding assay

This entry is part 2 of 1 in the series ELISA

ELISA-style-binding-assays (here shortly called ELISA) are very robust and relatively simple established assays. They are extremely useful for making single points measurements, and thereby testing a lot of proteins for interactions. With a little bit more effort, it’s also possible to extract proper binding constants.

With a small series of posts, I’d like to help with the most important steps in planning and executing an ELISA assay.

Here the most important pro and cons of an ELISA assay in a table. read more

Measuring pH dependent kOFFs easily with the BLItz interferometer

Recently we published a new paper, describing the detailed effect of various histidines for the very important pH dependent release of HSP47 (a collagen chaperone) in the golgi (see here for more background).

The initial technical difficulty for this project was to determine reliably, small differences in kOFF values at different pH. Measuring kOFF values is trivial, using a BIACORE, however, as the kOFF goes into your resulting curve twice (once during association as kobs and once in the dissociation) you can’t directly compare the curves by eye and need to trust the curve fittings. Although this is exactly what we did in the final paper, earlier in this project we convinced ourselves by a different assay design. read more

ELISA simulator

In preparation of a series about setting up ELISA-style-binding-assays, I programmed a little tool, which helps to predict ELISA curves.

If you have an idea about the KD you can easily determine how many datapoints (samples) at which concentration you need to get a well defined curve. You can also easily compare the effects of different serial dilution regimes and different hill coefficients.  Hopefully this helps to better plan your next ELISA style binding assay!

You can find it in the menu under >Tools< ore directly here. read more

A fresh start

Welcome to my little blog on BioAnalytics!

In this little blog I would like to share ideas, strategies and results around the general topic of protein-protein interactions and bioanalytical research on proteins. Some posts will be more directed to students or first-time-biochemists and these will be relatively instructional (like howtos and one side effect is that I can use them for my students). Other posts will be more directed towards experts and might be related to my actual research. I will try to publish something every Monday so stay tuned. read more