TITLE: Quantification of nonprotein sulfhydryl groups (NPSH) optimized for zebrafish brain tissue
AUTHORS: Adrieli Sachett, Matheus Gallas-Lopes, Greicy M M Conterato, Radharani , Ana Herrmann, Angelo Piato

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Zebrafish are incresingly used as a model animal in neuroscience research. Here we describe a protocol to quantify nonprotein sulfhydryl groups (NPSH), an indirect evaluation of the levels of reduced glutathione (GSH), a major oxidative stress defense in the central nervous system. </div></div>

[STEPS]
?. [Preparing the reagents]
The first step is to prepare the reagents to be used in the quantification of nonprotein sulfhydryl groups (NPSH);
?. [Preparing the reagents]
5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) :    1.1.1 Weigh carefully  of DTNB in a piece of aluminum foil;    1.1.2 Transfer the DTNB to a beaker of appropriate size;    1.1.3 Add  of absolute ethanol to the beaker to dissolve the salt;    1.1.4 Transfer your solution to a  volumetric flask;    1.1.5 Using absolute ethanol, complete the solution's volume to reach ;    1.1.6 Store the solution in an amber flask of appropriate size covered with aluminum foil at ;
0.0396 g
9 mL
10 mL
10 mL
8 °C
?. [Preparing the reagents]
Potassium phosphate buffer :     1.2.1 Weigh  of monobasic potassium phosphate (KH2PO4) in a beaker of appropriate size;    1.2.2 Dissolve the salt with  of ultrapure water;    1.2.3 Transfer the solution to a  volumetric flask;    1.2.4 Using ultrapure water, complete the solution's volume to reach ;    1.2.5 Weigh  of dibasic potassium phosphate (K2HPO4) in a beaker of appropriate size;    1.2.6 Dissolve the salt with  of ultrapure water;    1.2.7 Transfer the solution to a  volumetric flask;    1.2.8 Mix both solutions slowly in a  beaker following the steps below;Transfer  of the monobasic potassium phosphate (KH2PO4) solution to the beaker;Use a pH sensor to evaluate your solution. Expected conditions:;            If the pH of your solution is lower than 7 adjust the pH adding drops of the dibasic potassium phosphate (K2HPO4) solution;            If the pH of your solution is above 7 adjust the pH adding drops of the monobasic potassium phosphate (KH2PO4) solution;After adjusting the pH of this initial solution, proceed to add, slowly, drops of both buffer solutions (monobasic potassium phosphate and dibasic potassium phosphate). Use Pasteur pipettes to add the solutions. Mix your solutions using a pH sensor, making sure the mix of both buffers is always at ;
13.609 g
90 mL
100 mL
100 mL
17.418 g
90 mL
100 mL
500 mL
50 mL
?. [Preparing the reagents]
Trichloroacetic acid (TCA) 6%:    1.3.1 Weigh  of TCA in a beaker of an appropriate size;    1.3.2 Dissolve the TCA with  of ultrapure water;    1.3.3 Transfer your solution to a  volumetric flask;    1.3.4 Using ultrapure water, complete the solution's volume to reach ;    1.3.5 Store this solution in an amber flask at  ;
6 g
50 mL
100 mL
100 mL
8 °C
?. [Deproteinizaton of your samples]
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;
?. [Deproteinizaton of your samples]
Prepare   microtubes, to be used to store the samples, with the correct information. The number of microtubes depends on the number of samples;
1.5 mL
?. [Deproteinizaton of your samples]
Before preparing your samples for deproteinization, you must calculate the sample volume that corresponds to of proteins. This calculation can be based on the Bradford method described elsewhere;    2.2.1 To estimate the volume of the sample corresponding to  of proteins, divide the amount of protein needed () 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
50 µg
50 µg
50 µg
?. [Deproteinizaton of your samples]
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 of 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);  AB1Sample (µL)TCA 6% (µL)2Depends on the volume of the sample corresponding to 50 µg  of proteins and the number of replicates of the same sample you are planning to evaluateDepends on the volume of the sample. You should add the same volume of TCA solution as the volume of the sample you are planning to use (1:1)
50 µg
AB1Sample (µL)TCA 6% (µL)2Depends on the volume of the sample corresponding to 50 µg  of proteins and the number of replicates of the same sample you are planning to evaluateDepends on the volume of the sample. You should add the same volume of TCA solution as the volume of the sample you are planning to use (1:1)
?. [Deproteinizaton of your samples]
Use a vortexer to mix the samples for ;
?. [Deproteinizaton of your samples]
Centrifuge the samples ;
Centrifuge: 10000 34, 5 min, 4 10
?. [Microplate preparation and absorbance reading]
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;
?. [Microplate preparation and absorbance reading]
Before start pipetting, each well of the microplate should be marked for sample identification;
?. [Microplate preparation and absorbance reading]
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 , so the TFK is used so that every solution reaches this volume (e.g.,  of the sample +  of the TFK solution);  ABC1Well of the plateSample (µL)TFK 1 M  (µL)2Control02453SamplesDepends on the volume of the sample corresponding to 50 µg  of proteins.Depends on the volume of the sample. Volume needed for the final solution in the well to reach 245 µL.
245
50
195
ABC1Well of the plateSample (µL)TFK 1 M  (µL)2Control02453SamplesDepends on the volume of the sample corresponding to 50 µg  of proteins.Depends on the volume of the sample. Volume needed for the final solution in the well to reach 245 µL.
?. [Microplate preparation and absorbance reading]
Read the absorbance of the samples at  in a microplate reader;
412
?. [Microplate preparation and absorbance reading]
After reading the absorbance of the samples, add, in the dark,  of the DTNB solution  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 ;
15
260
?. [Microplate preparation and absorbance reading]
Leave your microplate in a dark room to incubate at room temperature for ;
?. [Microplate preparation and absorbance reading]
Read the absorbance of the samples at  in a microplate reader;
412
?. [Calculating data and determinig results]
Prepare to analyze the results obtained after reading the absorbance of the samples;
?. [Calculating data and determinig results]
Calculate the mean absorbance of the control solution both before and after adding the DTNB;Δcontrol = (Mean absorbance after DTNB - Mean absorbance before DTNB)
?. [Calculating data and determinig results]
Calculate the mean absorbance of the samples both before and after adding the DTNB;Δsample = (Mean absorbance after DTNB - Mean absorbance before DTNB)
?. [Calculating data and determinig results]
Subtract the Δcontrol value from the Δsample value for each of the samples;Absorbance of the sample = (Δsample - Δcontrol)
?. [Calculating data and determinig results]
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
?. [Calculating data and determinig results]
Results should be expressed as µmol of SH groups;µmol SH = Moles SH x 1000000
?. [Calculating data and determinig results]
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])
?. [Calculating data and determinig results]
Final results are expressed as µmol SH/mg protein.