Category: Automation and High-Throughput Technologies
Determining total protein concentration is an accepted means of comparing and standardizing biological samples. The utility of protein concentration assessment extends to high throughput screening platforms including proteomic and genomic applications, but ideally the protein concentration determination step should also be amenable to high throughput. But since most protein determination assays use absorbance measurement detection, it is much hard to minimize these assays to smaller, higher-throughput formats, unlike fluorescence or luminescence assays. Previously, it has been reported that some colorimetric assays, including the bicinchoninic acid (BCA) protein assay, can be performed in white plates using fluorescence detection. Exploiting their inherent fluorescence, white plates enable accurate protein determination in low volumes. Here, we show a comparison of two low volume 384-well plates (Greiner and Corning) and a 1536-well plate (Labcyte). Excitation / emission scanning of each plate with the CLARIOstar® microplate reader showed reasonably similar spectra for each plate with excitation maxima in the range of 435-450 and emission maxima in the range of 460-510. Furthermore, we found that both excitation and emission peaks were suppressed in the presence of BCA reagent and further decreases were observed in the presence of increasing protein concentrations following color development. Based on this spectral data the CLARIOstar® LVF monochromator was set to 435-15 for excitation and 562-20 for emission and the linear variable dichroic was automatically adjusted to 497.2 to read replicates of various concentrations of BSA (125-2000 mg/mL). Based on these results, transformed standard curves were graphed showing that the 12 ml (2 ml protein) samples in 384-well plates conform to a linear fit as do the 9 ml (1.5 ml protein) samples in 1536-well plates. Our results indicate that the BCA assay can be successfully miniaturized in all plate formats tested, to conserve valuable samples and decrease the amount of reagent used. Furthermore, using this approach, higher throughput can be achieved in protein concentration determination for subsequent analyses.
Carl Peters– Senior Applications Scientist, BMG Labtech, Cary, NC
Senior Applications Scientist
Dr. Carl Peters is a microplate reader Senior Application Scientist with BMG LABTECH. He obtained a PhD in Cell and Molecular Biology from Northwestern University while studying Protein Kinase C signaling. He also has a B.S. in Biology from Hastings College. Prior to BMG LABTECH, he was an adjunct or assistant professor of Biology, Biochemistry and Molecular Biology at several different universities.