Tuesday, April 3, 2018

Smaller is Better


Sample volume becomes increasingly critical as sample throughput increases for many common applications. Many of these applications, such as protein profiling using LC-MS/MS or RT-PCR, sequencing and micro-array analysis of nucleic acids, rely heavily on accurately determined molecular concentrations of purified biomolecules. Current biomolecular isolation protocols and kits result in yields ranging from nanogram to milligram amounts and are typically eluted in 10-100 μL volumes. Therefore, typical concentrations can range from sub ng/μL to thousands of ng/μL.

Quantification of these purified samples is routinely accomplished by spectrophotometric analysis at 280 nm for proteins and at 260 nm for nucleic acids in a UV transparent vessel. Measurements have traditionally been made in quartz cuvettes with a fixed pathlength of 1 cm and are typically associated with high precision and accurate measurements. Using standard 1 cm pathlength cuvettes, however, often requires diluting nucleic acid samples above about 100-200 ng/μL and protein samples above about 4 mg/mL. This can be less than ideal given the small volume of sample available. Microplates are used to determine analyte concentrations in a more condensed format of 96 samples per plate and lower sample volume. However, a variable pathlength dependent on well diameter and sample volume must be considered during determinations.


More recently, BioTek introduced the use of a micro-volume plate for rapid measurement of multiple undiluted samples with volumes as low as 2 μL, each with 0.5 mm nominal pathlength. The micro-volume plate also provides the capability to make measurements using a cuvette, and/or two vertical 1 cm pathlength vertical cells.

Take3

We have also shown the analytical performance of the plate for micro-volume protein analysis using either native protein absorbance at 280 nm or with the aid of the signal enhancing reagent bicinchoninic acid (BCA). Additionally, we have shown the ability of the accessory to perform micro-volume analysis using UV absorption for both dsDNA and RNA as well the use of the signal enhancing reagent PicoGreen™. Additional methods include monitoring cell growth using turbidity measurements for several cell types including mammalian and yeast cells.

Growth curves of JM109 depicting hourly A600 measurements using either a) 2 µL sample on Take3 accessory plate or b) 500 uL in a low-volume cuvette.

Further information, including a recent webinar highlighting the various methods, can be found at BioTek.com. Under the Applications section, look for Nucleic Acid Quantification or Total Protein Quantification for detailed information. The Resources section contains available on-demand webinars.

By: BioTek Instruments, Peter J. Brescia Jr., MSc, MBA

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