ML162- RNase Inhibitor · 2021. 1. 21. · ML106-100ML ML106-500 ML 500 ml Introduction: Bradford...

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1 Bradford Reagent Kit Packing Product Name Bradford Reagent 100 ml Product Code ML106-100ML ML106-500 ML 500 ml Introduction: Bradford Reagent is formulated for rapid and accurate quantitative estimation of protein samples by following the Bradford assay method which was developed by Marion M. Bradford in 1976. This reagent is conveniently supplied as ready-to-use form and no dilution is required while performing the assay. Description: Bradford Reagent contains a dye, Coomassie brilliant Blue G-250, which has an absorbance maximum of 465 nm in unbound state. The Bradford protein assay is based upon the formation of complexes between Coomassie Brilliant Blue G-250 and the protein samples in solution. When the protein sample binds to the dye the color of the solution turns blue from brown and there is a shift in the absorption maximum of the dye from 465 nm to 595 nm. This dye binding procedure is completed within 5 minutes and the blue colored complex formed is stable for one hour. Thus concentration of unknown protein sample can be derived by plotting its absorbance value on the standard curve. The standard curve is obtained from the absorbance readings of the series of standard protein dilutions assayed along side the unknown sample. Application: Bradford reagent is mainly used for the quantitative estimation of protein samples by the Bradford assay method. Composition: Bradford reagent consists of Coomassie Brilliant Blue G-250 in phosphoric acid and ethanol. Properties: Appearance : Brown colored solution Clarity : Clear and free of particles Suitability test : This solution has been tested and is suitable for use in quantitative estimation of protein samples by Bradford assay Storage conditions: Bradford Reagent has to be stored at 2 - 8 o C Quality control: Bradford Reagent is tested for quantitative estimation of unknown protein samples. Estimation of unknown protein using standard curve (with BSA) 1. Take seven test tubes and label them as Blank and 1 to 6. 2. Make dilutions of standard protein (BSA) with concentrations of 20, 16, 12, 8, 4 μg/200 μl by transferring respective amount of BSA solution (stock: 1mg/ml) and adjusting it to a total volume of 200 μl by adding distilled water as mentioned in the following table. 3. Add 1ml of Bradford’s Reagent to each test tube and mix the contents of each tube thoroughly by vortexing the tubes and incubate at room temperature (below 30 o C) for 10 minutes. 4. Transfer the content of the tubes to cuvettes and measure the absorbance at 595 nm wavelength. The information contained herein is believed to be accurate and complete. However no warranty or guarantee whatsoever is made or is to be implied with respect to such information or with respect to any product, method or apparatus referred to herein Tel: 00-91-22-6147 1919 Fax: 6147 1920, 2500 5764 Email : [email protected] Web : www.himedialabs.com A-516, Swastik Disha Business Park, Via Vadhani Indl. Est., LBS Marg, Mumbai - 400 086, India 23, Vadhani Industrial Estate,LBS Marg, Mumbai - 400 086, India. Tel. : (022) 4017 9797 / 2500 1607 Fax : (022) 2500 2286 Commercial Office Registered Office : WHO GMP CERTIFIED 15 Product Information Unzipping Genes

Transcript of ML162- RNase Inhibitor · 2021. 1. 21. · ML106-100ML ML106-500 ML 500 ml Introduction: Bradford...

Page 1: ML162- RNase Inhibitor · 2021. 1. 21. · ML106-100ML ML106-500 ML 500 ml Introduction: Bradford Reagent is formulated for rapid and a ccurate quantitative estimation of protein

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Bradford Reagent

Kit Packing Product Name Bradford Reagent 100 ml

Product Code ML106-100ML ML106-500 ML 500 ml

Introduction: Bradford Reagent is formulated for rapid and accurate quantitative estimation of protein samples by following the Bradford assay method which was developed by Marion M. Bradford in 1976. This reagent is conveniently supplied as ready-to-use form and no dilution is required while performing the assay.

Description: Bradford Reagent contains a dye, Coomassie brilliant Blue G-250, which has an absorbance maximum of 465 nm in unbound state. The Bradford protein assay is based upon the formation of complexes between Coomassie Brilliant Blue G-250 and the protein samples in solution. When the protein sample binds to the dye the color of the solution turns blue from brown and there is a shift in the absorption maximum of the dye from 465 nm to 595 nm. This dye binding procedure is completed within 5 minutes and the blue colored complex formed is stable for one hour. Thus concentration of unknown protein sample can be derived by plotting its absorbance value on the standard curve. The standard curve is obtained from the absorbance readings of the series of standard protein dilutions assayed along side the unknown sample.

Application: Bradford reagent is mainly used for the quantitative estimation of protein samples by the Bradford assay method.

Composition: Bradford reagent consists of Coomassie Brilliant Blue G-250 in phosphoric acid and ethanol.

Properties: Appearance : Brown colored solution

Clarity : Clear and free of particles

Suitability test : This solution has been tested and is suitable for use in quantitative estimation of protein

samples by Bradford assay

Storage conditions: Bradford Reagent has to be stored at 2 - 8 oC

Quality control: Bradford Reagent is tested for quantitative estimation of unknown protein samples.

Estimation of unknown protein using standard curve (with BSA)

1. Take seven test tubes and label them as Blank and 1 to 6.

2. Make dilutions of standard protein (BSA) with concentrations of 20, 16, 12, 8, 4 μg/200 μl by

transferring respective amount of BSA solution (stock: 1mg/ml) and adjusting it to a total volume of

200 μl by adding distilled water as mentioned in the following table.

3. Add 1ml of Bradford’s Reagent to each test tube and mix the contents of each tube thoroughly by

vortexing the tubes and incubate at room temperature (below 30oC) for 10 minutes.

4. Transfer the content of the tubes to cuvettes and measure the absorbance at 595 nm wavelength.

The information contained herein is believed to be accurate and complete. However no warranty or guarantee whatsoever is madeor is to be implied with respect to such information or with respect to any product, method or apparatus referred to herein

Tel: 00-91-22-6147 1919Fax: 6147 1920, 2500 5764

Email : [email protected] : www.himedialabs.com

A-516, Swastik Disha Business Park, Via Vadhani Indl. Est., LBS Marg, Mumbai - 400 086, India

23, Vadhani Industrial Estate,LBS Marg, Mumbai - 400 086, India. Tel. : (022) 4017 9797 / 2500 1607 Fax : (022) 2500 2286

Commercial Office Registered Office :

WHO GMP

CERTIFIED

15

P r o d u c t I n f o r m a t i o nUnz i p p i n g G en e s

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5. Plot a Standard Curve of absorbance at 595 nm on “Y” axis versus concentration of protein μg/200μl on “X” axis.

6. Record the value “x” of unknown sample from graph corresponding to the absorbance reading.

Fig: Standard curve for protein estimation by Bradford assay

Protein concentration can be calculated using following formula:

Concentration of Unknown in “μg”

Protein Concentration in Unknown Sample = ---------------------------------------- x 1000 μg/ml

Volume of sample in “μl”

96-well plate assay:

This assay should be performed in a 96-well plate. It is very fast and easy to quantitate multiple protein samples at a time. NOTE: For Standard Graph, 5 µl of 0.2-1.4mg/ml of protein sample can be used.

Protocol:

1. Prepare protein standards as mentioned above2. Add 5µl of standard protein samples to each well of 96 plate.3. Add 5 µl of unknown samples to the wells.

Tube No. Blank 1 5 4 6 2 3 Conc. of BSA (μg)

0.0 4 8 12 16 20 Unknown sample

Amt of Stock (μl) 0.0 4 8 12 16 20

200 μl Amt of diluent (μl) 200 196 192 188 184 180

Amt of Bradford Reagent (ml)

1 1 1 1 1 1 1

Vortex the tubes and incubate at RT for 10 minutes

Absorbance at 595 nm

Standard used: BSA

Please refer disclaimer Overleaf.

Page 3: ML162- RNase Inhibitor · 2021. 1. 21. · ML106-100ML ML106-500 ML 500 ml Introduction: Bradford Reagent is formulated for rapid and a ccurate quantitative estimation of protein

4. To each well, add 250µl of Bradford reagent.5. Mix well by pipetting or keep it in a shaker for 15-30 seconds.6. Incubate the samples for 5-45 minutes at room temperature and measure the absorbance at 595nm.7. The absorbance readings must be taken before 60 minutes or within 10 minutes after the completion

of incubation.8. Plot a Standard Curve of absorbance at 595 nm on “Y” axis versus concentration of

protein on “X” axis.9. Record the value “x” of unknown sample from graph corresponding to the absorbance reading and

determine the protein concentration.

Technical Assistance: At HiMedia we pride ourselves on the quality and availability of our technical support. For any kind of technical assistance, mail at [email protected].

PIML106_0/0719 ML106-00

Disclaimer :

User must ensure suitability of the product(s) in their application prior to use. Products conform solely to the information contained in thisand other related HiMedia™ publications. The information contained in this publication is based on our research and development workand is to the best of our knowledge true and accurate. HiMedia™ Laboratories Pvt Ltd reserves the right to make changes to specificationsand information related to the products at any time. Products are not intended for human or animal diagnostic or therapeutic use but forlaboratory, research or further manufacturing use only, unless otherwise specified. Statements contained herein should not be consideredas a warranty of any kind, expressed or implied, and no liability is accepted for infringement of any patents.

HiMedia Laboratories Pvt. Ltd. A-516,Swastik Disha Business Park,Via Vadhani Ind. Est., LBS Marg, Mumbai-400086, India. Customer care No.: 022-6147 1919 Email: [email protected] Website: www.himedialabs.com

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Do not use if package is damaged

Storage temperature

15°C

25°C

HiMedia Laboratories Pvt. Limited, 23 Vadhani Industrial Estate, LBS Marg,Mumbai-86,MS,India


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