Protein quantification

Protein quantification

High accuracy, low volume protein quantification

Techniques which require tedious one-by-one measurements or diluting down concentrated samples can eat up time and precious protein while simultaneously introducing significant error. UV/Vis spectroscopy on Big Lunatic and Stunner equips you with high-throughput total protein quantification at low volumes, without any additional reagents. Protein quantification on Big Lunatic and Stunner is spot-on with an accuracy within 2% and precision within 1% over a dynamic range from low to high concentrations: for ~1 to >150 mg/mL for a monoclonal antibody (Figure 1).

Figure 1. Measured absorbance for a dilution series of a monoclonal antibody up to 200 mg/mL.

Proteins absorb UV light with a peak at 280 nm. The absorbance value at this wavelength, or A280, is directly proportional to the pathlength through the sample, l, and the protein concentration, c. This relationship is described by Beer’s law, also called the Beer-Lambert law.

 

If known fixed pathlengths are used, then calculating concentration from the measured A280 is just missing one last piece of the puzzle: the molar extinction coefficient, ε.

A protein’s molar extinction coefficient depends on aromatic amino acids (Trp, Tyr and Phe) and Cys-Cys disulfide bonds. Post-translation modifications and protein confor­mation can also have an impact, as can buffer type, ionic strength and pH. A theoretical extinction coefficient can be calculated from the number of tryptophan, tyrosine, and cysteine residues in the amino acid sequence using the following equation:

 

When you combine this extinction coefficient with a measured A280 value and a fixed pathlength, you can measure protein concentration to crazy good levels of accuracy and precision (Figure 2).

Figure 2. Known vs. measured concentration averages and %CV for an IgG dilution series.

Big Lunatic and Stunner deliver high-quality protein quantification with A280 measurements from 0.03–275 OD without dilution for up to 96 samples on a plate. With the Fundamentals certified tryptophan standards, you can show off Big Lunatic and Stunner’s impressive linearity, high accuracy and low %CV, or check out this app note for examples of quantifying more proteins.

For those proteins which lack aromatic amino acids, Lunatic and Stunner are fully equipped for colorimetric protein quantification techniques like the bicinchoninic acid (BCA), Bradford or Lowry assays.

UV/Vis spectroscopy is one of the oldest and most well-established protein quantification techniques. Unchained Labs pushes UV/Vis to the next level with innovative optics, well-controlled consumables, and honed analytical techniques.

Unchained Labs UV/Vis instruments deliver:

  • Accurate and precise full-spectrum UV/Vis absorbance results from 230–750 nm
  • Results from tiny 2 µL sample volumes
  • Wide dynamic range from 0.03 to 275 OD
  • IgG quantification from 0.02–200 mg/mL
  • 96 UV/Vis reads in about 10 minutes
  • Microfluidic circuits to protect samples from evaporation and cross-contamination
  • Fixed pathlength microcuvettes for maximum sensitivity and reproducibility
  • Purpose-built applications for protein quantification of purified or non-purified samples
  • Low maintenance and no clean-up
  • Accuracy you can show off with the Fundamentals tryptophan standards
  • Performance Verification for USP and Ph. Eur. compliance

Big Lunatic

Big Lunatic makes batch quantification of protein a no-brainer. All you need is 2 µL and 10 minutes to measure up to 96 samples. Run them straight-up, even at high concentrations, without ever diluting. Measurements are spot-on with precision within 1% and accuracy within 2% and are fully compliant with USP and Ph. Eur. guidelines. The SBS plate-based format means integrating with lab automation and liquid handlers is a snap. Big Lunatic gets biologics UV/Vis quantification on the money every time. Just drop, load, and read.

Stunner

Stunner combines Big Lunatic’s plate-based, high-throughput UV/Vis accuracy and precision with dynamic light scattering for a one-of-a-kind combination. Pairing up these techniques checks concentration, hydrodynamic size, polydispersity, and detection of aggregates off your to-do list in one shot. Sync up sample quantity and quality your way.

Stunner

Little Lunatic

Little Lunatic is there for labs that have fewer samples and lower requirements. It uses a chip-based version of the Big Lunatic plate to get quick concentration measurements of up to 16 samples in 5 minutes. Researchers that just need a quick check of concentration can get what they need from this little guy and its glove-friendly touch screen.

Little Lunatic

More info

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