TITLE: Protein quantification  protocol optimized for zebrafish brain tissue (Bradford method)
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 the total amount of proteins in zebrafish brain tissue.</div></div>

[STEPS]
?. [Preparing the reagents]
The first step is to prepare the reagents to be used in protein quantification;
?. [Preparing the reagents]
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  of Coomassie Brilliant Blue;    1.1.2 Dissolve completely the Coomassie Brilliant Blue in  of absolute ethanol in a beaker of appropriate size;    1.1.3 Add  of 85% orthophosphoric acid to the solution;    1.1.4 Transfer your solution to a  volumetric flask;    1.1.5 Using ultrapure water, complete the solution's volume to reach ;    1.1.6 Store the solution in a glass flask covered in aluminum foil;
0.05 g
25 mL
50 mL
500 mL
500 mL
?. [Preparing the reagents]
Albumin solution: The dilution of this solution depends on the concentration needed to build your standard curve (in this case );    Preparing a stock solution of :       1.2.1 Weigh  of bovine albumin;    1.2.2 Dissolve completely the albumin powder in  of ultrapure water using a beaker of appropriate size;    1.2.3 Transfer your solution to a  volumetric flask;    1.2.4 Using ultrapure water, complete the solution's volume to reach ;    1.2.5 Stock this solution at , in samples of  or  using plastic microtubes;    1.2.5 Unfreeze one of those  samples and dilute it to the concentration needed () following the calculation below:C1 x V1 = C2 x V2x V1 =  x V1 =   of the stock solution ()    1.2.6 Using a micropipette, collect  os the stock solution and transfer it to a  volumetric flask;    1.2.7 Using ultrapure water, complete the solution's volume to reach ;
0.1 g
9 mL
10 mL
10 mL
-20 °C
1.5 mL
2 mL
10 mL
1 mL
1000
1000
10 mL
10 mL
?. [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;
?. [Preparing the reagents]
Phosphate buffered saline solution (PBS): You should also prepare a PBS solution () as you will need  of the solution at room temperature to determine the control point of the standard curve.
10 µl
?. [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  , 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;
?. [Microplate preparation and protein quantification]
Before start pipetting, each well of the microplate should be marked for sample identification. Absorbance should be read no later than  after pipetting tissue samples;
?. [Microplate preparation and protein quantification]
Use one of the microtubes with the bovine albumin solution to generate the standard curve to quantify the proteins in your tissue ;
?. [Microplate preparation and protein quantification]
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;  ABCD1Well/Point of the curveBradford reagentPBSBovine albumin (1 mg/mL)2Control190 μL10 μL-31198 μL-2 μL42196 μL-4 μL53194 μL-6 μL64192 μL-8 μL75190 μL-10 μL86188 μL-12 μL97186 μL-14 μL108184 μL-16 μL
ABCD1Well/Point of the curveBradford reagentPBSBovine albumin (1 mg/mL)2Control190 μL10 μL-31198 μL-2 μL42196 μL-4 μL53194 μL-6 μL64192 μL-8 μL75190 μL-10 μL86188 μL-12 μL97186 μL-14 μL108184 μL-16 μL
?. [Microplate preparation and protein quantification]
Read the absorbance of at  in a microplate reader;
595
?. [Microplate preparation and protein quantification]
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;
ABCD1Well/Point of the curveAbsorbance 1Absorbance 2Mean2Control0.2620.2580.260310.5030.5110.507420.5770.5890.583530.7250.7200.723640.8520.8970.875750.8760.9040.890
?. [Microplate preparation and protein quantification]
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  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;  ABC1Well/Point of the curveBradford reagentTissue sample2Sample 1190 µL10 µL3Sample 2190 µL10 µL4Sample 3190 µL10 µL5Sample (n)190 µL10 µL
ABC1Well/Point of the curveBradford reagentTissue sample2Sample 1190 µL10 µL3Sample 2190 µL10 µL4Sample 3190 µL10 µL5Sample (n)190 µL10 µL
?. [Microplate preparation and protein quantification]
Read the absorbance of the samples at  in a microplate reader;
595
?. [Microplate preparation and protein quantification]
A demonstration of the expected results for the absorbances of the tissue samples is shown below;
ABCDEFG1Well/Point of the curveAbs 1Abs 2Abs3Abs 4MeanCorrected mean absorbance of the sample2Sample 11.1001.0381.1111.056--3Sample 21.1281.0601.0371.057--4Sample 30.7870.7840.7370.8240.7690.5095Sample 40.9450.8410.8760.8510.8560.5966Sample 51.0090.9691.0010.9500.9600.7007Sample 60.7271.0871.0270.464--8Sample 71.1481.1401.1191.139--9Sample 81.0291.0160.9561.026--10Sample 91.0681.0000.9910.9910.9910.73111Sample 100.4440.8140.8850.8910.8880.62812Sample 111.1041.0551.0271.019--13Sample 121.2101.2181.1961.188--14Sample 131.1670.7630.8390.8290.8340.57415Sample 140.8420.7260.7400.7500.7390.479
?. [Calculating data and determinig 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 factorsor MCF  = _FC1+ FC2 + FC3 + FC4 + FC5_        5(number of factors)
Using a solution of   of bovine albumin and the expected results shown above:  ABCD1Well/Point of the curveAbsorbance 1Absorbance 2Mean2Control0.2620.2580.260310.5030.5110.507420.5770.5890.583530.7250.7200.723640.8520.8970.875750.8760.9040.890Correction factors:Well/point of the curve: 1Mean absorbance of the point - Control absorbance = 0.507 - 0.260 = 0.247[  ] Albumin:     1 mg – 1000 µL                             x     –    2 µL                              x = 2 µgCF1 = 2 µg / 0.247 = 8.09717 µg/nmWell/point of the curve: 2Mean absorbance of the point - Control absorbance = 0.583 - 0.260 = 0.323[  ] Albumin:     1 mg – 1000 µL                             x     –    4 µL                              x = 4 µgCF1 = 4 µg / 0.323 = 12.38390 µg/nmWell/point of the curve: 3Mean absorbance of the point - Control absorbance = 0.723 - 0.260 = 0.463[  ] Albumin:     1 mg – 1000 µL                             x     –    6 µL                              x = 6 µgCF1 = 6 µg / 0.463 = 12.95896 µg/nmWell/point of the curve: 4Mean absorbance of the point - Control absorbance = 0.875 - 0.260 = 0.615[  ] Albumin:     1 mg – 1000 µL                             x     –    8 µL                              x = 8 µgCF1 = 8 µg / 0.615 = 13.00813 µg/nmWell/point of the curve: 5Mean absorbance of the point - Control absorbance = 0.890 - 0.260 = 0.630[  ] Albumin:     1 mg – 1000 µL                             x     –    10 µL                              x = 10 µgCF1 = 10 µg / 0.630 = 15.87302 µg/nmMean correction factor:8.09717 + 12,38390 + 12,95896 + 13,00813 + 15,87302 = 62.32118 µg/nmMCF = 62,32118 / 5 = 12.464236 µg/nm
?. [Calculating data and determinig results]
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 );Amount of protein = _____(Corrected mean absorbance x MCF)______                                        10µLP.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)                                        10µLP.S. the dilution factor depends on the proportion of the dilution applied: DF (1:2) = 2DF (1:3) = 3DF (1:4) = 4
10
ABCDEFG1Well/Point of the curveAbs 1Abs 2Abs3Abs 4MeanCorrected mean absorbance of the sample2Sample 11.1001.0381.1111.056--3Sample 21.1281.0601.0371.057--4Sample 30.7870.7840.7370.8240.7690.5095Sample 40.9450.8410.8760.8510.8560.5966Sample 51.0090.9691.0010.9500.9600.7007Sample 60.7271.0871.0270.464--8Sample 71.1481.1401.1191.139--9Sample 81.0291.0160.9561.026--10Sample 91.0681.0000.9910.9910.9910.73111Sample 100.4440.8140.8850.8910.8880.62812Sample 111.1041.0551.0271.019--13Sample 121.2101.2181.1961.188--14Sample 131.1670.7630.8390.8290.8340.57415Sample 140.8420.7260.7400.7500.7390.479Calculating 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 proteinsSample 4:(0.596 x 12.464236) / 10 = 0.743     Dilution factor (1:2): 0.743 x 2 = 1.486 µg/mL of proteinsSample 5:(0.700 x 12.464236) / 10 = 0.872     Dilution factor (1:2): 0.634 x 2 = 1.745 µg/mL of proteinsSample 9:(0.731 x 12.464236) / 10 = 0.911 µg/mL of proteinsSample 10:(0.628 x 12.464236) / 10 = 0.783 µg/mL of proteinsSample 13:(0.574 x 12.464236) / 10 = 0.715 µg/mL of proteinsSample 14:(0.479 x 12.464236) / 10 = 0.597 µg/mL of proteins