Microplate Assays for Protein Quantitation

Quantitating protein samples is an important step before gel electrophoresis or Western blot analysis, and for measuring bound versus free protein levels in protein binding assays. The choice of assay format is often based on other components in the sample mixture. NanoOrange is compatible with reducing agents and is sensitive enough that many contaminants can be diluted out below threshold levels. CBQCA is compatible with lipoproteins or lipid protein mixtures.

CBQCA Protein Quantitation Kit

The CBQCA Protein Detection Kit accurately detects from 10 ng to 150 mg of protein, lipoprotein or lipid-protein mixtures in 100 to 200 µL. The reagent is essentially non-fluorescent in aqueous solution but reacts with protein to produce highly fluorescent derivatives.

Quant-iT Protein Assay Kit

The Quant-iT Protein Assay Kit makes protein quantitation easy and accurate, it is highly selective for protein and exhibits very little protein-to-protein variation. Common contaminants, such as salts, solvents, or DNA—but not detergents—are well tolerated in the assay which is performed at room temperature, and the signal is stable for 3 hours.

NanoOrange Protein Quantitation Kit

NanoOrange Protein Quantitation Kit provides an ultrasensitive assay for measuring the concentration of proteins in solution with accurate detection between 100 ng/mL and 10 mg/mL using a single dye concentration. The assay is compatible with reducing agents and nucleic acids do not interfere with protein quantitation. Given the sensitivity of the assay, common contaminants such as salts and detergents can generally be diluted below their threshold of interference.

cbqca-s001046
Detection of bovine serum albumin (BSA) using the CBQCA Protein Quantitation Kit. The primary plot shows detection of BSA from 50 ng to 1000 ng. Inset A shows that the detection range can extend up to 150 μg. Inset B shows that the lower detection limit can extend down to 10 ng. Each point is the average of four determinations.

Selection guide

 CBQCA Protein Quantitation KitQuant-iT Protein Assay KitNanoOrange Protein Quantitation Kit, 200-2000 Assays
TargetProteinProteinProtein
ReporterCBQCAQuant-iT Protein ReagentNanoOrange
Ex/Em (nm)465/550 nm470/570 nm485/590 nm
AssayQuantitation of proteins in the presence of lipids, membrane fractions or detergentsQuantitation of proteins in solutionQuantitation of proteins in solution
UsageDetects at concentrations between 10 ng/mL and 150 ng/mL in 200 µL assay volumeDetects in the range of 0.25–500 µg of protein in 200 µL assay volumeDetects at concentrations between 10 ng/mL and 10 µg/mL in 200 µL assay volume
ComponentsIncludes all reagents to prepare stock solutionIncludes assay buffer and standardsIncludes assay buffer and standards
Format300–800 assays (at 100–200 µL per assay)1,000 assays, with a 200 µL assay volume2,000 assays, with a 200 µL assay volume
Protocol outline
  1. Apply protein sample to microplate.
  2. Add KCN solution.
  3. Add CBQCA solution.
  4. Incubate for one hour.
  5. Measure fluorescence.
  6. Quantitate protein from standard curve.
  1. Load microplate with working solution.
  2. Add samples, mix well.
  3. Incubate at room temperature for 15 min.
  4. Measure fluorescence.
  5. Quantitate protein by comparing to standard curve.
  1. Combine samples with NanoOrange working solution in a microcentrifuge tube.
  2. Incubate sample at 90–95°C for 10 min.
  3. Cool and transfer to a microplate well.
  4. Measure fluorescence.
  5. Quantitate protein by comparing to standard curve.
Cat. No.C6667Q33210N6666

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High-sensitivity fluorescence detection for 6-1,536 samples can be quickly performed on the Varioskan ALF or Varioskan LUX Multimode Microplate Reader using Invitrogen reagents to enable optimal detection. Take advantage of automatic dynamic range selection to get optimal gain settings for each individual well and automation capabilities for even higher throughput.

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