The protein is known as the "building blocks of life" and is one of the most important macromolecules in life science. Proteins are polypeptides made up of amino acids and play various key roles in all aspects of biology. Protein quantitation is a very common practice for life scientists.
Simple, direct and automation-ready procedures for measuring protein concentration are very desirable. Protein assay kit is based on an improved Coomassie Blue G method. The dye forms a blue complex specifically with protein, and the intensity of color, measured at 595nm, is directly proportional to the protein concentration in the sample. The optimized formulation substantially reduces interference by substances in the raw samples and exhibits increased sensitivity towards peptides.
Applications:Direct Assays: total protein concentration.
Key Features:Sensitive and accurate. Use 10ul samples. Detection range 0.06–1.0mg /mL protein in 96-well plate assay
Simple and high-throughput. The “mix-and-read” procedure involves addition of a single working reagent and reading the optical density. Can be readily automated as a high-throughput assay in 96-well plates for thousands of samples per day
Low interference. Glucose, Tris, vitamins, and amino acids, DNA, RNA, salts, EDTA (< 12mM), phenol (< 50mM), urea (< 0.6 M), Triton (< 0.1%) and SDS (< 0.1% SDS) do not interfere in the assay
Versatility: assays can be executed in 96-well plate or cuvet.
Kit Contents: (500 tests in 96-well plates)Reagent: 20ml 5 x concentrateProtein standard: 1ml 1.0 mg/mL BSAStorage conditions. The kit is shipped at room temperature. Store the reagent at 4°C and standard at -20°C, respectively. Shelf life: 12 months after receipt
Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.
Procedures:Reagent Preparation:Prepare enough working reagent by adding 1 vol of the 5 x Reagent to 5 vol of distilled water. Bring reagent to room temperature before use
Procedure using 96-well plate:1. Dilute standard as shown in the Table. Transfer 10ul diluted Standards and diluted sample in duplicate wells of a clear bottom 96-well plate. Store diluted standards at -20°C for future use
No Premix+H2O Vol (ul) BSA (mg/mL) BSA (ug/10ul)1 100ul+0ul 100 1.0 102 80ul+20ul 100 0.8 83 60ul+40ul 100 0.6 64 40ul+60ul 100 0.4 45 30ul+70ul 100 0.3 36 20ul+80ul 100 0.2 27 10ul+90ul 100 0.1 18 0ul+100ul 100 0 02. Add 200ul working reagent and tap lightly to mix
3. Measure OD at 570-630nm (peak 595nm)
Procedure using cuvette:1. Prepare standards as in the 96-well plate assay. Transfer 50ul diluted Standards and 50ul samples to cuvets
2. Add 1000ul working reagent and tap lightly to mix
3. Measure OD at 570-630nm (peak 595nm).
Calculation:Subtract blank OD (water, #8) from the standard OD values and plot the OD against standard concentrations. Use the standard curve to determine the sample protein concentration.
Materials Required, But Not Provided:Pipeting devices and accessories
Procedure using 96-well plate:Blank 96-well plates (e.g. Corning Costar)
Plate reader for 96-well plate
Procedure using cuvette:Cuvets and spectrophotometer.
General Considerations:If protein concentration is > 1 mg/mL, dilute samples in distilled water, and use OD values that lie within the calibration curve to calculate the sample protein concentration. Reading can be performed as soon as the reagent and sample are mixed. High sensitivity can be achieved by adding 50ul sample to 200ul Reagent (detection range 3–200 ug/mL).
