Reduction of Nonspecific Binding of Yeast Protein to Grinding Beads
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|The use of mechanical disruption, such as
bead beating with glass beads, to homogenize cells, tissue, and small organisms is very
popular. By mixing roughly equal volumes of beads and sample, followed by
agitation (using either a vortexer or mixer mill), near complete lysis of
cells can be obtained. Nucleic acids for PCR analysis, proteins for gel
analysis, and enzymes for activity assays can all be liberated from cells
with only minor modifications to the same protocol. However, bead beating
small samples destined for quantitative analysis has a hidden problem that
can dramatically affect the accuracy and linearity of tests used on those
Biochemicals nonspecifically adsorb to glass and plastics. It is an inherent problem related to surface chemistry. When dealing with large quantities of cell or tissue homogenates, nonspecific binding is not a huge problem due to the excess of solutes in the sample. A small portion of solutes binds to available surfaces and “block” the open areas causing a minimal loss of the lysate. However, if a small sample is homogenized then the portion lost to nonspecific binding can be very significant. As the assays used to measure liberated molecules can be quite sensitive, as with real-time PCR and fluorescent-based enzyme assays, the loss of significant amounts of analyte to nonspecific binding can greatly skew data. To reduce nonspecific binding during homogenization, OPS Diagnostics developed Low Binding grinding beads that are coated via a proprietary process so as to minimize nonspecific binding of molecules.
To demonstrate the effectiveness of Low Binding grinding beads (both zirconium and glass beads), the yeast Saccharomyces uvarum was used as a test organism for homogenization. S. uvarum produces the periplasmic enzyme α-galactosidase when cultured on galactose-based medium, such as YPG (1% yeast extract, 2% peptone, 2% galactose). Cells were cultured overnight, washed, and then homogenized in microfuge tubes using a vortexer and Acid Washed or Low Binding beads. A total of six bead types were used to homogenize the yeast, namely 200 μm Zirconium (Acid Washed and Low Binding), 400 μm Silica (Acid Washed and Low Binding), and 800 μm Silica (Acid Washed and Low Binding). Following homogenization, samples were centrifuged and supernatant assayed for α-galactosidase using p-nitrophenyl- α-D-galactoside as a substrate. The hydrolysis of the α-glycolytic bond liberates p-nitrophenol, a yellow compound that can be measured spectrophotometrically. Figure 1 compares the supernatant enzyme activities generated from the six bead types.
Figure 1. Relative α-galactosidase activities from the homogenates generated using three sizes of Acid Washed and Low Binding silica and zirconium grinding beads.
With all bead types, the Low Binding beads yielded the highest enzyme activity. These beads are identical except for the coating process, thus differences in activity between Acid Washed and Low Binding is attributed to nonspecific binding of enzyme to the beads. The yields vary with bead type with Low Binding beads yielding 14%, 41%, and 45% more enzyme activity than Acid Washed beads for the 400 μm, 200 μm, and 800 μm beads, respectively. The results also demonstrate that bead size and type also impact overall yields of solutes during homogenization. Larger beads are often used for fungi and pollen while bacteria require smaller beads (100 μm beads, not shown, are often used for bacterial cells and spores).
Although yeast was used for this study, Low Binding Zirconium beads have been successfully used to homogenize bacteria collected from aquatic samples for real-time PCR analysis. The Low Binding property of the beads allowed for a lower detection limit as nucleic acids liberated during homogenization were less likely to adsorb to the beads, thus allowing for their amplification. In both cases, Low Binding grinding beads increased the sensitivity of the assay where a limited number of analytes were generated during the homogenization process