Navigation Links
Benchmark Plus Microplate Reader: Quantitation of Protein Concentration Using Two Different Colorimetric Assay Kits, Rev A

Theresa Redila-Flores, Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547 USA


The Bradford and Lowry assays are 2 colorimetric assays that are commonly used to quantitate protein concentration. Bio-Rads protein assay (catalog #500-0001), based on the Bradford dye-binding assay (Bradford 1976), measures total protein concentration. The standard assay is used for protein concentrations ranging from 50 g/ml to 1.0 mg/ml. This assay is based on the change of color of Coomassie Brilliant Blue G-250 dye with different protein concentrations, in which the dye binds to basic and aromatic amino acid residues. This assay is measured between 465 and 595 nm. Bio-Rads detergent compatible (DC) protein assay (catalog #500-0111) is similar to the Lowry assay (Lowry et al. 1951), but only requires a 15 min incubation. The reaction between protein and copper in the alkaline solution and the Folin reagent reduced by the copper-treated protein leads to color development. This protein assay is measured between 650 and 750 nm, with protein concentrations ranging from 200 g/ml to 1.0 mg/ml.


Procedure
Bradford Method (Bio-Rad Protein Assay) Using Clear 96- and 384-Well Plates
1. Dilute dye reagent with 1 part of dye reagent concentrate and 4 parts of distilled water. Filter through a Whatman #1 filter. The diluted solution is good for about 2 weeks when stored at room temperature.

2. Serially dilute standard bovine IgG protein 1:2, ranging from 31 g/ml to 1.0 mg/ml.

3. Aliquot 10 l of each diluted standard in triplicate into a clear 96-well microplate.

4. Add 200 l of diluted dye reagent. Mix thoroughly using a microplate mixer.

5. Incubate 5 min at room temperature.

6. Measure absorbance at 595 nm.

7. For a 384-well microplate, the protein standard and diluted dye reagent volumes are reduced to one quarter of the total volume used in a 96-well plate; that is, 50 l per well.

8. For 384-well measurements, aliquot 10 l of each diluted standard into a tube.

9. Add 200 l of diluted dye reagent. Vortex for about 5 sec.

10. Let incubate for 5 min at room temperature.

11. Aliquot 52.5 l per well. Make sure no bubbles are present, as this may cause reading errors.

12. Measure absorbance at 595 nm.


Lowry Method (Bio-Rad Detergent Compatible (DC) Protein Assay) using 96- and 384-Well Plates
1. Prepare working reagent as follows: Add 20 l of reagent S to 1 ml of reagent A as needed for assay.

2. Serially dilute the standard bovine IgG protein 1:2, from 125 g/ml to 2.0 mg/ml.

3. Aliquot 30 l of each of the diluted standards into a separate 2 ml tube.

4. Add 150 l of the working reagent to each tube and then add 1.2 ml of reagent B.

5. Vortex for about 5 sec.

6. Incubate at room temperature for 15 min.

7. Aliquot 230 l of each sample in triplicate into a clear 96-well microplate.

8. Measure absorbance at 750 nm.

9. For a 384-well microplate, aliquot 6.25 l of diluted standard into a 1.5 ml microfuge tube.

10. Add 31.25 l of working reagent and 250 l of reagent B.

11. Vortex for about 5 sec.

12. Incubate at room temperature for 15 min.

13. Aliquot 57.5 l of each sample in triplicate into a clear 384-well microplate.

14. Measure at 750 nm.


Results
Bio-Rad's protein assay kit gave linear results using the Benchmark Plus microplate reader to quantitate protein
concentration. The linear range for Bio-Rads protein assay was between 50 g/ml and 1.0 mg/ml in both 96- and 384-well plates (Figures 1 and 2). Readings were linear within their designated ranges with a correlation coefficient of ≥0.99.

Bio-Rad's detergent compatible (DC) protein assay kit gave linear results using the Benchmark Plus microplate reader to quantitate protein concentration. The linear range for both plates was between 125 g/ml and 2.0 mg/ml in both 96- and 384-well plates (Figures 3 and 4). Readings were linear within their designated ranges with a correlation coefficient of ≥0.99.


Conclusion
The Benchmark Plus microplate reader is an excellent instrument to quantitate concentrations of protein samples
using 2 of Bio-Rads protein assay kits. The correlation coefficients of all the graphs were ≥0.99, within the specified detection ranges of 31.25 g/ml to 1.0 mg/ml. Assays of both
96- and 384-well plate formats gave linear graphs, thus allowing a choice of using these 2 different plate formats with excellent results along with high throughput. The results
showed that the Benchmark Plus microplate reader exhibits excellent linearity, repeatability, and accuracy for use in 96- and 384-well plate formats.


References
Bradford MM, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem 72, 248254 (1976)

Lowry OH et al., Protein measurement with the Folin phenol reagent, J Biol Chem 193, 265275 (1951)


Coomassie is a trademark of Imperial Chemical Industries PLC.


back to top
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Benchmark Plus Microplate Reader Scan Well Feature
2. Cell Proliferation Assay Using the Benchmark Plus Microplate Spectrophotometer
3. Cerenkov Counting Performance on the TopCountTM Microplate Scintillation and Luminescence Counter
4. Counting Non-Aqueous Samples in the TopCount Microplate Scintillation and Luminescence Counter
5. Fluorometric Protease Assays in the SpectraMax Gemini Microplate Spectrofluorometer: Example Using Caspase3 (MaxLine Application Note #35)
6. LIVE/DEAD Viability/Cytotoxicity Assay for Animal Cells Using the SpectraMax Gemini XS Fluorescence Microplate Reader (MaxLine Application Note #43)
7. Using the OligoGreen Oligonucleotide Quantitation Reagent in the Gemini XS, Gemini EM and SpectraMax M2 Microplate Readers (MaxLine Application Note #21)
8. PathCheck Applied to Measurement of Protein Solutions in the SpectraMax Plus Microplate Spectrophotometer (MaxLine Application Note #26)
9. Microdetermination of Phosphorus Using the SpectraMax Plus Microplate Spectrophotometer: Choice of Microplate, Cuvette, or Test Tube Assay Formats (MaxLine Application Note #24)
10. UV Absorbance Measurements in SpectraMax Microplate Spectrophotometers MaxLine Application Note #32)
11. Ca++-Induced G Protein Coupled Receptor (GPCR) Activation Monitored via Aequorin Luminescence in the LMax Microplate Luminometer (MaxLine Application Note #42)
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/27/2016)... 27, 2016  Global demand for enzymes is ... 2020 to $7.2 billion.  This market includes enzymes ... products, biofuel production, animal feed, and other markets) ... biocatalysts). Food and beverages will remain the largest ... consumption of products containing enzymes in developing regions.  ...
(Date:6/27/2016)... ... June 27, 2016 , ... Parallel 6 , ... announced today the Clinical Reach Virtual Patient Encounter CONSULT module which enables ... the physician and clinical trial team. , Using the CONSULT module, patients and physicians ...
(Date:6/27/2016)... 27, 2016   Ginkgo Bioworks , a leading ... was today awarded as one of the World ... world,s most innovative companies. Ginkgo Bioworks is engineering ... real world in the nutrition, health and consumer ... with customers including Fortune 500 companies to design ...
(Date:6/24/2016)... NC (PRWEB) , ... June 24, 2016 , ... Researchers ... the most commonly-identified miRNAs in people with peritoneal or pleural mesothelioma. Their findings are ... to read it now. , Diagnostic biomarkers are signposts in the blood, lung ...
Breaking Biology Technology:
(Date:4/15/2016)... -- A new partnership announced today will help life ... a fraction of the time it takes today, ... insurance policies to consumers without requiring inconvenient and ... rapid testing (A1C, Cotinine and HIV) and higi,s ... pulse, BMI, and activity data) available at local ...
(Date:3/31/2016)...   LegacyXChange, Inc. ... LegacyXChange is excited to release its first ... be launched online site for trading 100% guaranteed authentic ... also provide potential shareholders a sense of the value ... industry that is notorious for fraud. The video is ...
(Date:3/22/2016)... India , March 22, 2016 /PRNewswire/ ... market research report "Electronic Sensors Market for Consumer ... Proximity, & Others), Application (Communication & IT, ... Geography - Global Forecast to 2022", published ... industry is expected to reach USD 26.76 ...
Breaking Biology News(10 mins):