Portelite™ Fluorimetric Protein Quantitation Kit *Optimized for CytoCite™ and Qubit™ Fluorometers*

Protocol summary

1. Prepare and add BSA standards or test samples (10 µL)
2. Prepare and add Prolite™ Orange working solution (190 µL) in a 0.2 mL PCR tube (Cat# CCT100)
3. Incubate at room temperature for 15 minutes
4. Monitor fluoroscence with CytoCite™ or Qubit fluorometer

Important notes
Bring all the kit components at room temperature before starting the experiment.

Prolite™ Orange working solution:
Add 5 µL of Prolite™ Orange (200X) (Component A) to 995 µL of Sample Dilution Buffer (Component E) and mix them well. Note: Do not mix the working solution in a glass container. Note: Prepare the amount as needed.

This protocol is generated based upon Qubit^® Fluorometer.

Run protein assay

1. Add 190 µL/well of Prolite™ Orange working solution into each tube.
   
   
2. Add 10 µL BSA standards (Component B, C, D) or 10 µL samples into the 190 µL Prolite™ Orange working solution tube to make the final assay volume 200 µL/tube.
   
   
3. Incubate the reaction at room temperature for 15 minutes. Note: Protect the samples from light and avoid holding the samples in hands.
   
   
4. Insert the samples into CytoCite™ and monitor the fluorescence with Green fluorescent channel. Follow the procedure appropriate for CytoCite™ Fluorometer. See the link below for detailed instructions:
   https://devices.aatbio.com/documentation/user-manual-for-cytocite-fluorometer

Brief protocol for Qubit^® fluorometer

1. Press Protein on the Home screen of the Qubit^® Home screen and proceed to press Read standards.
   
   
2. Insert each of the 3 tubes contains standards into the sample chamber.
   
   
3. Close the lid and press Read standards.
   
   
4. The instrument displays the results and generates calibration curve.
   
   
5. Press Run samples and select sample volume to 10 µL.
   
   
6. Insert the sample tube into the sample chamber.
   
   
7. Close the lid and press Read tube.
   
   
8. The instrument displays the results on the assay screen. The top value is the original sample concentration and bottom value is the diluted concentration.

PREPARATION OF STANDARD SOLUTION

For CytoCite™ Fluorometer assays, you have the choice to run a calibration with your own protein standards. Here is a brief protocol to generate a customized protein standard curve.

1. Prepare a protein solution of 400µg/ml (400 ng/µL) in PBS buffer.
   
   
2. Perform 1:2 serial dilution with PBS buffer to get 200, 100, 50, 25, 12.5 ng/µl serial standard dilutions.
   
   
3. Add 190 µL of Prolite™ Orange working solution into a 0.2 mL PCR tube.
   
   
4. Add 10 µL standards or 10 µL samples into each tube.
   
   
5. Incubate the reaction at room temperature for 15 minutes.
   
   
6. Insert the samples into CytoCite™ and monitor the fluorescence with Green fluorescent channel.