Different microplates, different results!

When analyzing biological data in the microplate format, usually three items come into play: the microplate, the assay kit, and the microplate reader. However, when the assay does not work properly or does not give the expected results, the blame is quite often put on the microplate reader. Probably the fact that it is the last ring of the chain or the most expensive item makes it easier to use it as a scapegoat.

Dr Tobias Pusterla Dr Tobias Pusterla (10)

Nevertheless, sometimes it is the most unexpected ring of that chain that plays the major role: the microplate. A paper published last year in the scientific journal Analytical Biochemistry (Anal Biochem. 2014 February 1; 446: 44–52) provides an interesting example. In this paper, Cui et al. tested the proteasome activity in different types of microplates from different manufacturers. The researchers came up with different results, showing that the performance of the proteasome assay is affected by the properties of the microplates.

Fig 1: Different Microplates

Apart from colour, three main types of microplates are available: non-binding or low-binding, medium-binding, and high-binding, with “normal” polystyrene microplates, usually referred to as medium-binding.

In this paper, four different types of 96-well black polystyrene microplates were compared: one non-binding, two medium-binding (different manufacturers), and one high-binding.

The most common method for proteasome activity determination is the incubation of a sample with a fluorescent proteasome substrate, with or without a specific inhibitor, in a black microplate. Fluorescent 7-amino-4-methylcoumarin (AMC) is a quite common label for this purpose. As the excitation and emission spectra of bound and unbound AMC are different, this allows the free AMC released by proteolytic cleavage to be easily detected with a fluorometer.

Proteasome activity is usually determined using standard curves, as the determination of the specific activity of the proteasome depends on the amount of AMC released in a given time per unit of protein. In this paper, already standard curves obtained measuring free AMC showed significant differences between all four plate types. Fluorescence measurement of free AMC showed that a medium binding plate from a specific manufacturer gave the highest fluorescence, whereas the high binding plate gave the lowest signal.

Interestingly, while the source of assay kits, proteins, chemicals, and instruments are usually stated in peer-reviewed publications, the microplate source is often neglected.


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