Protein quantification  protocol optimized for zebrafish brain tissue (Bradford method)

Adrieli Sachett; Matheus Gallas-Lopes; Greicy M M Conterato; Radharani; Ana Herrmann; Angelo Piato

Sep 08, 2020

Protein quantification protocol optimized for zebrafish brain tissue (Bradford method)

DOI

dx.doi.org/10.17504/protocols.io.bjnfkmbn

¹Universidade Federal do Rio Grande do Sul;
²Universidade Federal de Santa Catarina

Fish behavior and physiology
LAPCOM

Angelo Piato

Universidade Federal do Rio Grande do Sul

DOI: dx.doi.org/10.17504/protocols.io.bjnfkmbn

Protocol Citation: Adrieli Sachett, Matheus Gallas-Lopes, Greicy M M Conterato, Radharani Benvenutti Benvenutti, Ana P Herrmann, Angelo Piato 2020. Protein quantification protocol optimized for zebrafish brain tissue (Bradford method). protocols.io https://dx.doi.org/10.17504/protocols.io.bjnfkmbn

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: August 12, 2020

Last Modified: September 08, 2020

Protocol Integer ID: 40359

Keywords: Protein quantification, Zebrafish brain tissue,

Abstract

Zebrafish are incresingly used as a model animal in neuroscience research. Here we describe a protocol to quantify the total amount of proteins in zebrafish brain tissue.

Guidelines

This protocol is intended to standardize protein quantification of zebrafish brain tissue samples. It can be adapted for other fish species.

Materials

MATERIALS
ReagentGloves 
Reagent96 well plate 
ReagentSurgical mask 
ReagentMicropipette (0.5 - 10 μL) 
ReagentMicropipette (100 - 1000 μL) 
Reagent13x045 mm  needles 
ReagentMultichannel pipette (5 μL; 30-300 μL) 
ReagentSynergy™ HTX Multi-Mode Microplate ReaderBiotek 
STEP MATERIALS
ReagentBovine albumin fraction VINLABCatalog #1870 
Reagentortho-Phosphoric acid 85%Sigma-aldrichCatalog #1005732500 
ReagentBrilliant Blue GSigma-aldrichCatalog #27815 
ReagentEthanolMerck MilliporeCatalog #100983 
ReagentPhosphate buffered saline powder, pH 7.4, for preparing 1 L solutions Millipore SigmaCatalog #P3813 

Protocol materials

ReagentPhosphate buffered saline powder, pH 7.4, for preparing 1 L solutions Merck MilliporeSigma (Sigma-Aldrich)Catalog #P3813 
ReagentMicropipette (0.5 - 10 μL) 
Reagent13x045 mm  needles 
ReagentBovine albumin fraction VINLABCatalog #1870 
Reagentortho-Phosphoric acid 85%Merck MilliporeSigma (Sigma-Aldrich)Catalog #1005732500 
ReagentGloves 
ReagentBrilliant Blue GMerck MilliporeSigma (Sigma-Aldrich)Catalog #27815 
ReagentEthanolMerck Millipore (EMD Millipore)Catalog #100983 
ReagentSurgical mask 
ReagentMicropipette (100 - 1000 μL) 
ReagentMultichannel pipette (5 μL; 30-300 μL) 
ReagentSynergy™ HTX Multi-Mode Microplate ReaderBiotek 
Reagent96 well plate 
ReagentBrilliant Blue GMerck MilliporeSigma (Sigma-Aldrich)Catalog #27815 
ReagentEthanolMerck Millipore (EMD Millipore)Catalog #100983 
Reagentortho-Phosphoric acid 85%Merck MilliporeSigma (Sigma-Aldrich)Catalog #1005732500 
ReagentBovine albumin fraction VINLABCatalog #1870 
ReagentPhosphate buffered saline powder, pH 7.4, for preparing 1 L solutions Merck MilliporeSigma (Sigma-Aldrich)Catalog #P3813 

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

Preparing the reagents

The first step is to prepare the reagents to be used in protein quantification;

Bradford reagent: Prepare and use this reagent under dim or no light, making sure all glassware used is covered in aluminum foil to avoid photodegradation of the reagent. Prepare and use this reagent at room temperature;
    1.1.1 Weigh Amount0.05 g   of Coomassie Brilliant Blue;
ReagentBrilliant Blue GMerck MilliporeSigma (Sigma-Aldrich)Catalog #27815 
    1.1.2 Dissolve completely the Coomassie Brilliant Blue in Amount25 mL   of absolute ethanol in a beaker of appropriate size;
ReagentEthanolMerck Millipore (EMD Millipore)Catalog #100983 
    1.1.3 Add Amount50 mL   of 85% orthophosphoric acid to the solution;
Reagentortho-Phosphoric acid 85%Merck MilliporeSigma (Sigma-Aldrich)Catalog #1005732500 
    1.1.4 Transfer your solution to a Amount500 mL   volumetric flask;
    1.1.5 Using ultrapure water, complete the solution's volume to reach Amount500 mL  ;
    1.1.6 Store the solution in a glass flask covered in aluminum foil;

Albumin solution: The dilution of this solution depends on the concentration needed to build your standard curve (in this case Concentration1 mg/mL  );
    Preparing a stock solution of Concentration10 mg/mL  :   

    1.2.1 Weigh Amount0.1 g   of bovine albumin;
ReagentBovine albumin fraction VINLABCatalog #1870 
    1.2.2 Dissolve completely the albumin powder in Amount9 mL   of ultrapure water using a beaker of appropriate size;
    1.2.3 Transfer your solution to a Amount10 mL   volumetric flask;
    1.2.4 Using ultrapure water, complete the solution's volume to reach Amount10 mL  ;
    1.2.5 Stock this solution at Temperature-20 °C  , in samples of Amount1.5 mL   or Amount2 mL   using plastic microtubes;
    1.2.5 Unfreeze one of those Concentration10 mg/mL   samples and dilute it to the concentration needed (Concentration1 mg/mL  ) following the calculation below:

C1 x V1 = C2 x V2
Concentration10 mg/mL  x V1 = Concentration1.0 mg/mL   x Amount10 mL  
V1 = Amount1 mL    of the stock solution (Amount1000 µL  )
    1.2.6 Using a micropipette, collect Amount1000 µL   os the stock solution and transfer it to a Amount10 mL   volumetric flask;
    1.2.7 Using ultrapure water, complete the solution's volume to reach Amount10 mL  ;

Phosphate buffered saline solution (PBS): You should also prepare a PBS solution (Ph7.4)) as you will need Amount10 µL   of the solution at room temperature to determine the control point of the standard curve.
ReagentPhosphate buffered saline powder, pH 7.4, for preparing 1 L solutions Merck MilliporeSigma (Sigma-Aldrich)Catalog #P3813 

Microplate preparation and protein quantification

Use a conventional 96-well microplate to run your samples. Once again, protein quantification using Bradford reagent should be performed under dim or no light, making sure the microplate is carefully covered in aluminum foil to avoid photodegradation of the reagent. Also, this step should occur at room temperature;

Before start pipetting, each well of the microplate should be marked for sample identification. Absorbance should be read no later than Duration01:00:00   after pipetting tissue samples;

Use one of the microtubes with the bovine albumin solution to generate the standard curve to quantify the proteins in your tissue Go to  ;

To generate the standard curve, fill the wells of your microplate as described below. You should provide duplicates or triplicates of each point of the curve to make your quantification more precise. Using a micropipette fill the wells in this order: Bradford reagent, Phosphate buffered saline solution (PBS), and bovine albumin solution (mixing the solution with the pipette tip to homogenize the content of each well). Air bubbles should be perforated with a needle to avoid bias in the analysis;

Well/Point of the curveBradford reagentPBSBovine albumin (1 mg/mL)
Control190 μL10 μL-
1198 μL-2 μL
2196 μL-4 μL
3194 μL-6 μL
4192 μL-8 μL
5190 μL-10 μL
6188 μL-12 μL
7186 μL-14 μL
8184 μL-16 μL
 
Example of the configuration of the plate to determine the standard curve. CTRL: control.

Read the absorbance of at Amount595 nm   in a microplate reader;

A demonstration of the expected results for the absorbances of the samples composing the curve is shown below. Calculate the mean absorbance to use in the further quantification of brain protein content;
Expected result

Well/Point of the curveAbsorbance 1Absorbance 2Mean
Control0.2620.2580.260
10.5030.5110.507
20.5770.5890.583
30.7250.7200.723
40.8520.8970.875
50.8760.9040.890
Use duplicates or triplicates of each point to assure that your quantification is precise.
Example of the expected result in the plate. Different blue shades of blue are a result of the different quantities of protein present in each of the wells.

Following the determination of your standard curve, proceed to the quantification of your tissue samples. Using an adequate micropipette, fill the wells of your microplate as described below. You should provide triplicates or quadruplicates of each sample to make your quantification more precise. Using a micropipette fill the wells in this order: Firstly, fill the wells with the Bradford reagent followed by the tissue samples, mixing the solution with the pipette tip to homogenize the content of each well. Tissue sample collection and preparation are described elsewhere. The absorbance must be read in a maximum of Duration01:00:00   after pipetting the samples as stated above. Always use new tips for each sample and make sure that any researcher who handles the samples and plates is wearing a mask and gloves to avoid contamination. Air bubbles should be perforated with a needle to avoid bias in the analysis;
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

Well/Point of the curveBradford reagentTissue sample
Sample 1190 µL10 µL
Sample 2190 µL10 µL
Sample 3190 µL10 µL
Sample (n)190 µL10 µL
Example of the configuration of the plate to quantify the protein content of your sample.
 
 

Read the absorbance of the samples at Amount595 nm   in a microplate reader;

A demonstration of the expected results for the absorbances of the tissue samples is shown below;
Expected result

Well/Point of the curveAbs 1Abs 2Abs3Abs 4MeanCorrected mean absorbance of the sample
Sample 11.1001.0381.1111.056--
Sample 21.1281.0601.0371.057--
Sample 30.7870.7840.7370.8240.7690.509
Sample 40.9450.8410.8760.8510.8560.596
Sample 51.0090.9691.0010.9500.9600.700
Sample 60.7271.0871.0270.464--
Sample 71.1481.1401.1191.139--
Sample 81.0291.0160.9561.026--
Sample 91.0681.0000.9910.9910.9910.731
Sample 100.4440.8140.8850.8910.8880.628
Sample 111.1041.0551.0271.019--
Sample 121.2101.2181.1961.188--
Sample 131.1670.7630.8390.8290.8340.574
Sample 140.8420.7260.7400.7500.7390.479
What to look for when revising your data:

The corrected mean absorbance of the sample is calculated by: Mean absorbance - Mean absorbance of the control sample of the curve. 

Any absorbance reading above 1 (in italic) must be disregarded as the quantification won't be precise. If all of your readings are above 1, follow step 3.

When taking readings in triplicates or quadruplicates, one or two of the absorbance values can be disregarded if they are significantly different from the others (in bold and italic) and the average can be taken from the remaining values to ensure greater homogeneity of the data.
Example of the expected result in the plate. The blue color of the solution is directly related to the amount of protein present in each of the wells.

Protein quantification: optional steps

If the absorbance of your samples is above 1, the samples should be diluted in the portion of 1:2 (or even 1:3, 1:(n)) and go through a new reading phase as described above Go to   , always remembering to multiply the final absorbance value obtained by the dilution factor (as shown in the calculations below).

Calculating data and determinig results

Follow the calculations below to get your results;

Calculate the correction factor for your bovine albumin standard curve;

    4.1.1 Correction factor (CF): Subtract the absorbance value of the control point of the curve from the mean absorbance of the point you are calculating. Divide the concentration of albumin of the well/point of the curve by the resulting value from the subtraction before;

CF = _____[  ] Albumin____
           Mean Abs – Control Abs

    4.1.2 Mean correction factor (MCF): The mean correction factor is calculated by the arithmetic mean of the correction factors for each point of the curve;

MCF = ∑Correction factors / Count of correction factors

or 

MCF  = _FC1+ FC2 + FC3 + FC4 + FC5_
        5(number of factors)
Expected result
Using a solution of Concentration1 mg/mL   of bovine albumin and the expected results shown above:
Well/Point of the curveAbsorbance 1Absorbance 2Mean
Control0.2620.2580.260
10.5030.5110.507
20.5770.5890.583
30.7250.7200.723
40.8520.8970.875
50.8760.9040.890
Correction factors:
Well/point of the curve: 1
Mean absorbance of the point - Control absorbance = 0.507 - 0.260 = 0.247

[  ] Albumin:     1 mg – 1000 µL
                             x     –    2 µL                              x = 2 µg

CF1 = 2 µg / 0.247 = 8.09717 µg/nm

Well/point of the curve: 2
Mean absorbance of the point - Control absorbance = 0.583 - 0.260 = 0.323

[  ] Albumin:     1 mg – 1000 µL
                             x     –    4 µL                              x = 4 µg

CF1 = 4 µg / 0.323 = 12.38390 µg/nm

Well/point of the curve: 3
Mean absorbance of the point - Control absorbance = 0.723 - 0.260 = 0.463

[  ] Albumin:     1 mg – 1000 µL
                             x     –    6 µL                              x = 6 µg

CF1 = 6 µg / 0.463 = 12.95896 µg/nm

Well/point of the curve: 4
Mean absorbance of the point - Control absorbance = 0.875 - 0.260 = 0.615

[  ] Albumin:     1 mg – 1000 µL
                             x     –    8 µL                              x = 8 µg

CF1 = 8 µg / 0.615 = 13.00813 µg/nm

Well/point of the curve: 5
Mean absorbance of the point - Control absorbance = 0.890 - 0.260 = 0.630

[  ] Albumin:     1 mg – 1000 µL
                             x     –    10 µL                              x = 10 µg

CF1 = 10 µg / 0.630 = 15.87302 µg/nm

Mean correction factor:

8.09717 + 12,38390 + 12,95896 + 13,00813 + 15,87302 = 62.32118 µg/nm

MCF = 62,32118 / 5 = 12.464236 µg/nm

Calculate the amount of protein of each of your tissue samples (the results should be expressed as µg/mL of proteins);

    4.2.1 The amount of protein in your samples is calculated by multiplying the corrected mean absorbance of your sample to the mean corrected factor calculated above and dividing the result by the volume of the tissue sample used  (in this case Amount10 µL  );

Amount of protein = _____(Corrected mean absorbance x MCF)______
                                        10µL

P.S. remember the corrected mean absorbance of the sample is calculated by subtracting the absorbance value of the control point of the standard curve from the mean absorbance of the sample that you are calculating.

    4.2.2 If you are using diluted samples, remember to multiply the final absorbance value obtained by the dilution factor (DF).

Amount of protein = _____(Corrected mean absorbance x MCF)_____    x   (DF)
                                        10µL

P.S. the dilution factor depends on the proportion of the dilution applied: 

DF (1:2) = 2
DF (1:3) = 3
DF (1:4) = 4
Expected result

Well/Point of the curveAbs 1Abs 2Abs3Abs 4MeanCorrected mean absorbance of the sample
Sample 11.1001.0381.1111.056--
Sample 21.1281.0601.0371.057--
Sample 30.7870.7840.7370.8240.7690.509
Sample 40.9450.8410.8760.8510.8560.596
Sample 51.0090.9691.0010.9500.9600.700
Sample 60.7271.0871.0270.464--
Sample 71.1481.1401.1191.139--
Sample 81.0291.0160.9561.026--
Sample 91.0681.0000.9910.9910.9910.731
Sample 100.4440.8140.8850.8910.8880.628
Sample 111.1041.0551.0271.019--
Sample 121.2101.2181.1961.188--
Sample 131.1670.7630.8390.8290.8340.574
Sample 140.8420.7260.7400.7500.7390.479
Calculating the amount of protein in the available samples above: 
Sample 3:
(0.509 x 12.464236) / 10 = 0.634     Dilution factor (1:2): 0.634 x 2 = 1.269 µg/mL of proteins

Sample 4:
(0.596 x 12.464236) / 10 = 0.743     Dilution factor (1:2): 0.743 x 2 = 1.486 µg/mL of proteins

Sample 5:
(0.700 x 12.464236) / 10 = 0.872     Dilution factor (1:2): 0.634 x 2 = 1.745 µg/mL of proteins

Sample 9:
(0.731 x 12.464236) / 10 = 0.911 µg/mL of proteins

Sample 10:
(0.628 x 12.464236) / 10 = 0.783 µg/mL of proteins

Sample 13:
(0.574 x 12.464236) / 10 = 0.715 µg/mL of proteins

Sample 14:
(0.479 x 12.464236) / 10 = 0.597 µg/mL of proteins

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

Step 2.6

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

Well/Point of the curve	Bradford reagent	PBS	Bovine albumin (1 mg/mL)
Control	190 μL	10 μL	-
1	198 μL	-	2 μL
2	196 μL	-	4 μL
3	194 μL	-	6 μL
4	192 μL	-	8 μL
5	190 μL	-	10 μL
6	188 μL	-	12 μL
7	186 μL	-	14 μL
8	184 μL	-	16 μL

Well/Point of the curve	Absorbance 1	Absorbance 2	Mean
Control	0.262	0.258	0.260
1	0.503	0.511	0.507
2	0.577	0.589	0.583
3	0.725	0.720	0.723
4	0.852	0.897	0.875
5	0.876	0.904	0.890

Well/Point of the curve	Bradford reagent	Tissue sample
Sample 1	190 µL	10 µL
Sample 2	190 µL	10 µL
Sample 3	190 µL	10 µL
Sample (n)	190 µL	10 µL

Well/Point of the curve	Abs 1	Abs 2	Abs3	Abs 4	Mean	Corrected mean absorbance of the sample
Sample 1	1.100	1.038	1.111	1.056	-	-
Sample 2	1.128	1.060	1.037	1.057	-	-
Sample 3	0.787	0.784	0.737	0.824	0.769	0.509
Sample 4	0.945	0.841	0.876	0.851	0.856	0.596
Sample 5	1.009	0.969	1.001	0.950	0.960	0.700
Sample 6	0.727	1.087	1.027	0.464	-	-
Sample 7	1.148	1.140	1.119	1.139	-	-
Sample 8	1.029	1.016	0.956	1.026	-	-
Sample 9	1.068	1.000	0.991	0.991	0.991	0.731
Sample 10	0.444	0.814	0.885	0.891	0.888	0.628
Sample 11	1.104	1.055	1.027	1.019	-	-
Sample 12	1.210	1.218	1.196	1.188	-	-
Sample 13	1.167	0.763	0.839	0.829	0.834	0.574
Sample 14	0.842	0.726	0.740	0.750	0.739	0.479

Public workspaceProtein quantification protocol optimized for zebrafish brain tissue (Bradford method)

Protein quantification protocol optimized for zebrafish brain tissue (Bradford method)