The QuDye Protein Quantification kit includes all required reagents to quantitate protein except of deionized water. The range of initial protein concentration for measurements with Qubit™ fluorometer is 12.5-5000 µg/ml. The whole procedure is described step by step below:

Step 1.

Depending on the assay number in the kit it includes whether 10x concentrate or 1x ready-to-use Protein quantification buffer. If you have 1x solution, pass to the next step. If you have 10x concentrate buffer, prepare the 1x Protein quantification buffer by diluting the 10x concentrate 1:10 by deionized water.

View of QuDye Protein Quantification kit, QuDye Protein Quantification kit product view

You will need 200 μL of Protein quantification buffer for each of three standards and each sample. For instance, you are going to measure the protein concentration of 3 samples. In such a case you should prepare: 200 μL * 3 standards + 200 μL * 3 samples + 200 μL to include pipetting inaccuracies = 1400 μL of Protein quantification buffer. Mix 140 μL of 10x Protein quantification buffer concentrate  and 1260 μL of deionized water.

Step 2.

Prepare the QuDye working solution (only in plastic, never in glass tube) by diluting the 200x QuDye concentrate reagent 1:200 with 1x Protein quantification buffer (Use whether 1x ready-to-use Protein quantification buffer included in the kit, or diluted 10x buffer from Step 1). You will need about 200 μL of QuDye working solution for each of three standards and each sample.

Making the QuDye working solution, QuDye working solution preparation

For instance, you are going to measure the protein concentration of 3 samples. In such a case you should prepare following amounts of dye working solution: 200 μL * 3 standards + 200 μL * 3 samples + 200 μL to include pipetting inaccuracies = 1400 μL of QuDye working solution. Mix 1393 μL of 1х Protein quantification buffer and 7 μL of 200x QuDye reagent.

Step 3.

Prepare standards: mix 190 μL of prepared in Step 2 QuDye working solution and 10 μL of standard in thin-walled and optical-transparent 0.5 ml tube. In total you must prepare 3 standard solutions.

Prepare samples: mix 180-199 µL of prepared in Step 2 QuDye working solution and 20-1 µL of sample. To maintain accuracy avoid pipetting very small volumes while sample diluting. Initial sample concentration must be within the range from 12.5 μg/mL to 5000 µg/mL.

Vortex all tubes for 2-3 seconds and incubate the tubes for 15 minutes at room temperature.

Preparation of protein standards for quantitation, Pipeting protein standards for quantitationVortexing of samples and standards before quantitation, vortexing of protein

For instance, you need to measure the sample concentration in the range: 5000 µg/mL and 12.5 μg/mL. Dilute a sample with a concentration of 5000 µg/mL 200-fold: mix 199 µL of QuDye working solution and 1 µL of sample. The sample with a concentration of  12.5 μg/mL should be diluted 10-fold: mix 180 µL of QuDye working solution and 20 µL of sample. This approach will give the most accurate results of protein quantitation.

The next steps should be carried out according to the instructions to the fluorometer. Depending on the version of the fluorometer, the names of menu items may differ from specified below (specified for Qubit 1):

Step 4.

1) On the Home screen of the Qubit fluorometer, you should choose «Quant – iT Protein» as the assay type. Press «Go».

Home screen of Qubit (protein quanttation), Qubit home screen for protein quantitationSelecting Quant – iT Protein in Qubit menu, Qubit menu (protein quantitation)

2) With each preparation of the QuDye working solution, you must calibrate the fluorometer. Select "Run new calibration" and press "Go".

3) Insert the tube containing Standard #1 into the sample chamber, close the lid, then press "Go". When the reading is complete (~3 seconds), remove Standard #1. 

Insert the tube containing Standard #2 into the sample chamber, close the lid, then press"Go". When the reading is complete, remove Standard #2.

Insert the tube containing Standard #3 into the sample chamber, close the lid, then press"Go". When the reading is complete, remove Standard #3.

Calibrating Qubit fluorometer, Qubit calibrationInserting standard to qubit, Qubit calibration

4) Insert the tube containing your sample into the sample chamber, close the lid, then press "Go". On the screen you will see the QF value.

Calculate the concentration of the sample by the formula: Concentration of the sample = QF value x 200/sample volume.

Measuring protein sample with Qubit, Qubit protein measurementMeasuring sample with Qubit, Qubit protein quantitation

Qubit™ is trade mark of Thermo Fisher Scientific