Sep 16, 2021

Public workspaceQuantification of nonprotein sulfhydryl groups (NPSH) optimized for zebrafish brain tissue V.2

DOI
dx.doi.org/10.17504/protocols.io.bx8tprwn
  • 1Universidade Federal do Rio Grande do Sul;
  • 2UFRGS
  • LAPCOM
Angelo Piato
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DOI: dx.doi.org/10.17504/protocols.io.bx8tprwn
Protocol Citation: Adrieli Sachett, Radharani Benvenutti Benvenutti, Ana P Herrmann, Angelo Piato, Matheus Gallas-Lopes, Greicy M M Conterato 2021. Quantification of nonprotein sulfhydryl groups (NPSH) optimized for zebrafish brain tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.bx8tprwnVersion created by Matheus Gallas-Lopes
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: September 15, 2021
Last Modified: September 16, 2021
Protocol Integer ID: 53235
Keywords: NPSH, Oxidative stress, Zebrafish brain tissue,
Abstract
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.
Guidelines
This protocol is intended to standardize nonprotein sulfhydryl groups quantification of zebrafish brain tissue samples. It can be adapted for other fish species.
Materials
MATERIALS
ReagentGloves
Reagent96 well plate
ReagentEppendorf tubes 1.5 mL uncoloredEppendorf CentrifugeCatalog #022363204
ReagentMiniV ortexerVWR ScientificCatalog #58816-121
ReagentSurgical mask
ReagentMicropipette (0.5 - 10 μL)
ReagentMicropipette (100 - 1000 μL)
ReagentpH meter
ReagentCentrifuge 5424 REppendorfCatalog #5404000022
ReagentSynergy™ HTX Multi-Mode Microplate ReaderBiotek
STEP MATERIALS
Reagent55′-Dithiobis(2-nitrobenzoic acid)Sigma-aldrichCatalog #D8130
ReagentEthanolMerck MilliporeCatalog #100983
ReagentEthanolMerck MilliporeCatalog #100983
ReagentMonobasic potssium phosphateNUCLEARCatalog #318312
ReagentPotassium phosphate dibasic NeonCatalog #11361
ReagentTrichloroacetic acid (TCA)Sigma – AldrichCatalog #T6399
Protocol materials
Reagent55′-Dithiobis(2-nitrobenzoic acid)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D8130
ReagentEthanolMerck Millipore (EMD Millipore)Catalog #100983
ReagentMonobasic potssium phosphateNUCLEARCatalog #318312
ReagentPotassium phosphate dibasic NeonCatalog #11361
ReagentTrichloroacetic acid (TCA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #T6399
ReagentSurgical mask
ReagentGloves
Reagent96 well plate
ReagentEthanolMerck Millipore (EMD Millipore)Catalog #100983
ReagentMiniV ortexerVWR International (Avantor)Catalog #58816-121
ReagentEppendorf tubes 1.5 mL uncoloredEppendorfCatalog #022363204
ReagentpH meter
ReagentSynergy™ HTX Multi-Mode Microplate ReaderBiotek
ReagentMicropipette (0.5 - 10 μL)
ReagentMicropipette (100 - 1000 μL)
ReagentCentrifuge 5424 REppendorfCatalog #5404000022
Reagent55′-Dithiobis(2-nitrobenzoic acid)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D8130
ReagentEthanolMerck Millipore (EMD Millipore)Catalog #100983
ReagentEthanolMerck Millipore (EMD Millipore)Catalog #100983
ReagentMonobasic potssium phosphateNUCLEARCatalog #318312
ReagentPotassium phosphate dibasic NeonCatalog #11361
ReagentTrichloroacetic acid (TCA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #T6399
Safety warnings
Use personal protective equipment (including lab coat, masks, and gloves) when manipulating chemical and biological samples. Read the Safety Data Sheets of the reagents.
Before start
This protocol was standardized at LAPCOM (Psychopharmacology and Behavior Laboratory at UFRGS) to assess biochemical parameters in zebrafish brain tissue. Protocols you should read before proceeding with this method:
CITATION
Adrieli Sachett, Matheus Gallas-Lopes, Radharani Benvenutti, Greicy M M Conterato, Ana Herrmann, Angelo Piato. How to prepare zebrafish brain tissue samples for biochemical assays.
LINK
https://protocols.io/view/how-to-prepare-zebrafish-brain-tissue-samples-for-bjkdkks6

CITATION
Adrieli Sachett, Matheus Gallas-Lopes, Greicy M M Conterato, Radharani Benvenutti, Ana Herrmann, Angelo Piato. Optimized protein quantification protocol for zebrafish brain tissue (Bradford method).
LINK
https://protocols.io/view/optimized-protein-quantification-protocol-for-zebr-bjnfkmbn

Preparing the reagents
Preparing the reagents
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) Concentration10 mM :
1.1.1 Weigh carefully Amount0.0396 g of DTNB in a piece of aluminum foil;
Reagent55′-Dithiobis(2-nitrobenzoic acid)VWR InternationalCatalog #D8130
1.1.2 Transfer the DTNB to a beaker of appropriate size;

1.1.3 Add Amount9 mL of absolute ethanol to the beaker to dissolve the salt;
ReagentEthanolVWR InternationalCatalog #100983
1.1.4 Transfer your solution to a Amount10 mL volumetric flask;

1.1.5 Using absolute ethanol, complete the solution's volume to reach Amount10 mL ;
ReagentEthanolVWR InternationalCatalog #100983
1.1.6 Store the solution in an amber flask of appropriate size covered with aluminum foil at Temperature8 °C ;
Potassium phosphate buffer Concentration1 Molarity (M) :

1.2.1 Weigh Amount13.609 g of monobasic potassium phosphate (KH2PO4) in a beaker of appropriate size;
ReagentMonobasic potssium phosphateVWR InternationalCatalog #318312
1.2.2 Dissolve the salt with Amount90 mL of ultrapure water;

1.2.3 Transfer the solution to a Amount100 mL volumetric flask;

1.2.4 Using ultrapure water, complete the solution's volume to reach Amount100 mL ;

1.2.5 Weigh Amount17.418 g of dibasic potassium phosphate (K2HPO4) in a beaker of appropriate size;
ReagentPotassium phosphate dibasic VWR InternationalCatalog #11361
1.2.6 Dissolve the salt with Amount90 mL of ultrapure water;

1.2.7 Transfer the solution to a Amount100 mL volumetric flask;

1.2.8 Mix both solutions slowly in a Amount500 mL beaker following the steps below;
  • Transfer Amount50 mL of the monobasic potassium phosphate (KH2PO4) solution to the beaker;
  • Use a pH sensor to evaluate your solution. Expected conditions:Ph7.0 ;
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 Ph7.0;;

Trichloroacetic acid (TCA) 6%:

1.3.1 Weigh Amount6 g of TCA in a beaker of an appropriate size;
ReagentTrichloroacetic acid (TCA)VWR InternationalCatalog #T6399
1.3.2 Dissolve the TCA with Amount50 mL of ultrapure water;

1.3.3 Transfer your solution to a Amount100 mL volumetric flask;

1.3.4 Using ultrapure water, complete the solution's volume to reach Amount100 mL ;

1.3.5 Store this solution in an amber flask at Temperature8 °C ;

Deproteinizaton of your samples
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;
CITATION
Adrieli Sachett, Matheus Gallas-Lopes, Radharani Benvenutti, Greicy M M Conterato, Ana Herrmann, Angelo Piato. How to prepare zebrafish brain tissue samples for biochemical assays.
LINK
https://protocols.io/view/how-to-prepare-zebrafish-brain-tissue-samples-for-bjkdkks6

Prepare Amount1.5 mL 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 Amount50 µg of proteins. This calculation can be based on the Bradford method described elsewhere;
CITATION
Adrieli Sachett, Matheus Gallas-Lopes, Greicy M M Conterato, Radharani Benvenutti, Ana Herrmann, Angelo Piato. Optimized protein quantification protocol for zebrafish brain tissue (Bradford method).
LINK
https://protocols.io/view/optimized-protein-quantification-protocol-for-zebr-bjnfkmbn

2.2.1 To estimate the volume of the sample corresponding to Amount50 µg of proteins, divide the amount of protein needed (Amount50 µg ) 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 Amount50 µg 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);

Sample (µL)TCA 6% (µL)
Depends 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)

Use a vortexer to mix the samples for Duration00:00:10 ;
Centrifuge the samples Centrifigation10000 x g, 4°C, 00:05:00 ;
Centrifigation
Microplate preparation and absorbance reading
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;
Critical
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 Amount245 µL , so the TFK is used so that every solution reaches this volume (e.g., Amount50 µL of the sample + Amount195 µL of the TFK solution);


Well of the plateSample (µL)TFK 1 M (µL)
Control0245
SamplesDepends 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.

Read the absorbance of the samples at Amount412 nm in a microplate reader;
After reading the absorbance of the samples, add, in the dark, Amount15 µL of the DTNB solution Concentration10 mM 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 Amount260 µL ;

Leave your microplate in a dark room to incubate at room temperature for Duration01:00:00 ;
Incubation
Read the absorbance of the samples at Amount412 nm in a microplate reader;
Calculating data and determinig results
Calculating data and determinig results
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.
Citations
Adrieli Sachett, Matheus Gallas-Lopes, Radharani Benvenutti, Greicy M M Conterato, Ana Herrmann, Angelo Piato. How to prepare zebrafish brain tissue samples for biochemical assays
https://protocols.io/view/how-to-prepare-zebrafish-brain-tissue-samples-for-bjkdkks6
Adrieli Sachett, Matheus Gallas-Lopes, Greicy M M Conterato, Radharani Benvenutti, Ana Herrmann, Angelo Piato. Optimized protein quantification protocol for zebrafish brain tissue (Bradford method)
https://protocols.io/view/optimized-protein-quantification-protocol-for-zebr-bjnfkmbn
Step 2
Adrieli Sachett, Matheus Gallas-Lopes, Radharani Benvenutti, Greicy M M Conterato, Ana Herrmann, Angelo Piato. How to prepare zebrafish brain tissue samples for biochemical assays
https://protocols.io/view/how-to-prepare-zebrafish-brain-tissue-samples-for-bjkdkks6
Step 2.2
Adrieli Sachett, Matheus Gallas-Lopes, Greicy M M Conterato, Radharani Benvenutti, Ana Herrmann, Angelo Piato. Optimized protein quantification protocol for zebrafish brain tissue (Bradford method)
https://protocols.io/view/optimized-protein-quantification-protocol-for-zebr-bjnfkmbn