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 a 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 a 1x solution, pass it to the next step. If you have 10x concentrate buffer, prepare the 1x Protein quantification buffer by diluting the 10x concentrate 1:10 with deionized water.
You will need 200 μL of Protein quantification buffer for each of the 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 the 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 the 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 a 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. The initial sample concentration must be within the range of 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.
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 the 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 of the fluorometer. Depending on the version of the fluorometer, the names of menu items may differ from those specified below (specified for fluorometer 1):
Step 4.
1) On the Home screen of the fluorometer, you should choose «Quant – iT Protein» as the assay type. Press «Go».
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.
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.
