Amplite® Fluorimetric Protein Quantitation Kit *Orange Fluorescence*

Protocol summary

1. Prepare Prolite™ Orange working solution (50 µL)
2. Add BSA standards and/or test samples (50 µL)
3. Incubate at room temperature for 30 minutes
4. Read fluorescence intensity at Ex/Em = 485/590 nm

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

Protein Enhancer stock solution (500X):
Add 100 µL of Protein Enhancer Buffer (Component E) into the vial of Protein Enhancer (Component C). Note: 20 µL of Protein Enhancer stock solution (500X) is enough for make 10 mL working solution. Unused Protein Enhancer stock solution (500X) should be stored in single use aliquots at -20 ^oC.

1. Protein Enhancer working solution:
Add 20 µL of Protein Enhancer stock solution (500X) into 10 mL of Assay Buffer (Component D) and mix them well.

2. Prolite™ Orange Working Solution:
Add 10 µL of Prolite™ Orange (Component A) into 5 mL of Assay Buffer (Component D) and mix them well. Note: 5 mL of Prolite™ Orange working solution is enough for 1 plate. Prepare enough Prolite™ Orange working solution fresh for each experiment.

Table 1. Layout of BSA standards and test samples in a solid black 96-well microplate. BS = BSA standard (BS1-BS7); BL = blank control; TS = test sample.

Table 2. Reagent composition for each well

Protein assay

1. Add 50 µL of BSA standard, blank control, and test samples (See table 1 and 2) into a solid black 96-well plate. Note: For a 384-well plate, add 20 µL of samples.
   
   
2. Add 50 µL/well of Prolite™ Orange working solution into BSA standard, blank control, and test samples to make the total assay volume of 100 µL/well. Note: For a 384-well plate, add 20 µL of Prolite™ Orange working solution into each well.
   
   
3. Incubate the reaction for 10 to 30 minutes at 37 ^oC, protected from light.
   
   
4. Monitor the the fluorescence increase with a fluorescence plate reader at Ex/Em = 485/590 nm.