Amplite® Rapid Colorimetric Total Protein Thiol Quantitation Assay Kit

Important notes:
When stored properly, the kit components should be stable for six months. Do not freeze Spin Column (Component C). Warm all the components before run the required assays. 50 to 100 µg of protein sample is needed for determining the amount of thiol amount.

Prepare Sample Solution:

1. Adjust the volume of 50 to 100 µg of protein sample to 100 µL with Assay Buffer (Component B). Note: The protein sample should be in pH = 6.0 buffer and without DTT or other reagent containing free thiols.

Run Thiol Assay:

1. Add the protein sample to one vial of Thiol Blue™ (Component A).
   
   
2. Mix them well by repeatedly pipetting for a few times or vortex the vial for a few seconds.
   
   
3. Keep the reaction mixture at room temperature and rotate or shake for 30 - 60 minutes.

Prepare Spin Column for Sample Purification:

1. Invert the Spin Column (Component C) several times to resuspend the settled gel and remove any bubbles.
   
   
2. Snap off the tip and place the column in the Washing Tube (2 mL, Component D). Remove the cap to allow the excess packing buffer to drain by gravity to the top of the gel bed. If column does not begin to flow, push cap back into column and remove it again to start the flow. Discard the drained buffer, and then place the column back into the Washing Tube. However, centrifuge immediately if the column is placed into a 12 x 75 mm test tube (not provided).
   
   
3. Centrifuge for 1 min in a swinging bucket centrifuge at 1,000x g to remove the packing buffer. Discard the buffer.
   
   
4. Apply 1 mL Assay Buffer (Component B) to the column, let the buffer drain out by gravity, or centrifuge the column for 1 min to remove the buffer. Discard the buffer from the collection tube. Repeat this process for 3 - 4 times.
   
   
5. Centrifuge for 2 minutes in a swinging bucket centrifuge at 1,000x g to remove the reaction buffer. Discard the buffer. Note: Spin Column (Component C) can fit into 2 mL microcentrifuge tubes or 12 x 75 mm test tubes for sample collection during centrifugation. Use the 2 mL microtubes provided with the columns for the initial column equilibration step. Swinging bucket centrifuges capable of generating a minimum force of 1,000x g are suitable for Bio-Spin column use. The gravitational force created at a particular revolution speed is a function of the radius of the microcentrifuge rotor. Consult the swinging bucket centrifuge instruction manual for the information about conversion from revolutions per minute (RPM) to centrifugal or g-force. Alternatively, use the equation to calculate the speed in RPM required to reach the gravitational force of 1,000x g.
   RCF(g) = (1.12 x 10^-5) X (RPM)² X r
6. RCF = the relative centrifugal force, RPM = the speed of the rotor, r = the radius in centimeters measured from the center of the rotor to the middle of the Bio-Spin column

Purify Reaction Product:

1. Place the column in a clean Collecting Tube (1.5 mL, Component E). Carefully load the sample (100 µL) directly to the center of the column.
   
   
2. After loading the sample, add 10 µL Assay Buffer (Component B) to the top and centrifuge the column for 5 min at 1,000x g, and collect the solution into the collecting tube.

Run Absorption Spectra with 0.2 mL or 0.5 mL Quartz Cuvette:

1. Dilute the reaction product by 5-folds with Assay Buffer (Component B) depending on the cuvette size used and the absorbance reading. Note: The dilution factor doesn’t affect the final thiol quantitation result.
   
   
2. Measure the absorption spectrum from 250 to 750 nm, or only read the absorbance number at 280 nm and 680 nm.