Quantification of nonprotein sulfhydryl groups (NPSH) optimized for zebrafish brain tissue

The first step is to prepare the reagents to be used in the quantification of nonprotein sulfhydryl groups (NPSH);

5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) : 10mM:

1.1.1 Weigh carefully `0.0396g` of DTNB in a piece of aluminum foil;

1.1.2 Transfer the DTNB to a beaker of appropriate size;



1.1.3 Add `9mL` of absolute ethanol to the beaker to dissolve the salt;

1.1.4 Transfer your solution to a `10mL` volumetric flask;





1.1.5 Using absolute ethanol, complete the solution's volume to reach `10mL`;

1.1.6 Store the solution in an amber flask of appropriate size covered with aluminum foil at `8°C`;

Potassium phosphate buffer : 1Molarity (M):

1.2.1 Weigh `13.609g` of monobasic potassium phosphate (KH<sub>2</sub>PO<sub>4</sub>) in a beaker of appropriate size;

1.2.2 Dissolve the salt with `90mL` of ultrapure water;





1.2.3 Transfer the solution to a `100mL` volumetric flask;





1.2.4 Using ultrapure water, complete the solution's volume to reach `100mL`;





1.2.5 Weigh `17.418g` of dibasic potassium phosphate (K<sub>2</sub>HPO<sub>4</sub>) in a beaker of appropriate size;

1.2.6 Dissolve the salt with `90mL` of ultrapure water;





1.2.7 Transfer the solution to a `100mL` volumetric flask;





1.2.8 Mix both solutions slowly in a `500mL` beaker following the steps below;

Trichloroacetic acid (TCA) 6%:

1.3.1 Weigh `6g` of TCA in a beaker of an appropriate size;

1.3.2 Dissolve the TCA with `50mL` of ultrapure water;





1.3.3 Transfer your solution to a `100mL` volumetric flask;





1.3.4 Using ultrapure water, complete the solution's volume to reach `100mL`;





1.3.5 Store this solution in an amber flask at  `8°C`;

To proceed with the quantification of nonprotein sulfhydryl groups in your samples, you first have to deproteinize them following the steps below. Tissue sample collection and preparation are described elsewhere;

Prepare 1.5mL microtubes, to be used to store the samples, with the correct information. The number of microtubes depends on the number of samples;

Before preparing your samples for deproteinization, you must calculate the sample volume that corresponds to 50µgof proteins. This calculation can be based on the Bradford method described elsewhere;

2.2.1 To estimate the volume of the sample corresponding to `50µg` of proteins, divide the amount of protein needed (`50µg`) by the total amount of proteins in the sample quantified by the Bradford method (example below);

Volume of the sample needed for the assay (µL) = 50 µg / total amount of proteins in the sample µg/µL

For each tissue sample, fill the plastic microtubes as described below. You should provide duplicates or triplicates of each sample to make your quantification more precise. The sample volume corresponding to 50µgof proteins is the volume needed to fill one of the wells of the microplate, so if you are planning to evaluate your samples in duplicates fill the microtube with two times that volume, if evaluating in triplicates, three times that volume, and so on. Using a micropipette fill the tubes in this order: Sample and TCA solution (mixing the solution with the pipette tip to homogenize the content);

Use a vortexer to mix the samples for 0h 0m 10s;

Centrifuge the samples 10000x g,4°C;

Use a conventional 96-well microplate to run your samples. Reagents should be at room temperature. Pipetting of DTNB should be performed under dim or no light, making sure the microplate is carefully covered in aluminum foil to avoid photodegradation of the reagent;

Before start pipetting, each well of the microplate should be marked for sample identification;

Using an adequate micropipette, fill the wells of your microplate as described below. You should provide duplicates or triplicates of each sample as stated above. Using a micropipette fill the wells in this order: sample and TFK. The TFK volume depends on the volume of the sample. All wells should have a final volume of 245µL, so the TFK is used so that every solution reaches this volume (e.g., 50µL of the sample + 195µL of the TFK solution);

Read the absorbance of the samples at 412nm in a microplate reader;

After reading the absorbance of the samples, add, in the dark, 15µL of the DTNB solution 10mM to each well (control and samples) previously filled (mixing the solution with the pipette tip to homogenize the content of wells). The final volume of every well should reach 260µL;

Leave your microplate in a dark room to incubate at room temperature for 1h 0m 0s;

Read the absorbance of the samples at 412nm in a microplate reader;

Prepare to analyze the results obtained after reading the absorbance of the samples;

Calculate the mean absorbance of the control solution both before and after adding the DTNB;

Δcontrol = (Mean absorbance after DTNB - Mean absorbance before DTNB)

Calculate the mean absorbance of the samples both before and after adding the DTNB;

Δsample = (Mean absorbance after DTNB - Mean absorbance before DTNB)

Subtract the Δcontrol value from the Δsample value for each of the samples;

Absorbance of the sample = (Δsample - Δcontrol)

Determine the number of moles of the group sulfhydryl (SH) in the sample using the calculation below;

Moles of SH = Abssample x 0.00026 14.15 x 0.67

Results should be expressed as µmol of SH groups;

µmol SH = Moles SH x 1000000

Calculate the amount of sulfhydryl groups per milligrams of protein;

µmol SH/mg protein = µmol SH x 1000 (amount of proteins in the sample [50 µg in this case])

Final results are expressed as µmol SH/mg protein.