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Coomassie (Bradford) Protein Assay Kit

Coomassie (Bradford) Protein Assay Kit

  • Model: AI-BTLP01
  • Note: Price/availability/specifications subject to change without notice. Unless otherwise indicated, our catalog and customized products are for research use only and not intended for human or animal diagnostic or therapeutic use.

    Product Description

     

    Product introduction:

    Bradford method (coomassie brilliant blue method) is one of the most sensitive and rapid methods for protein determination.When Bradford dyeing liquid (coomassie brilliant blue G250) and protein under acid condition, the solution color from dark brown to blue, maximum absorption wavelength of 456 nm to 595 nm, absorbance value and the content of protein in a certain concentration range there is better linear relation, by measuring absorbance value size and contrast standard protein absorbance value, determine the protein concentration, realize the rapid determination of protein concentration, stability and high sensitivity.

    Product features:

    1. rapid, 10-20 samples only need 10 minutes to complete.

    2. stable, plus sample blending can be determined after 5 minutes, 1 hour of absorbance change less than 10%.

    3. minimum volume for the 10-20 ul, minimum measuring protein 50 ng.

    4. materials such as surfactant, glycerin, but the solution memory protein amino acid composition, the combination of various proteins and dye efficiency may have differences.

    5. economic and practical: are measured in the MPP, can greatly save the sample and reagent consumption.

    Product composition:

    The component of product

     

    BSA(2mg/ml)

    1ml

    Coomassie G-250 dye

    50ml

    Storage temperature:

    Bradford dyeing liquid 2-8ºC save, BSA protein standard - 20ºC.Valid for one year.

    Operation steps:

    1. Take appropriate amount of Bradford stain solution from refrigerator as required;Preheat the enzyme marker for 20 minutes.

    2. Take appropriate standard products as required, add deionized water and dilute them to 100ug/ml(2mg/ml of the original solution), and mix them.

    Note: in principle, the protein sample should be diluted in the same solution as the standard product.But for simplicity, you can dilute the standard with 0.9% NaCl or PBS.Protein standard can also be diluted - 20ºC long-term preservation.Deionized water is adopted by default in the following steps, and diluent can be selected according to the conditions.

    3 .Standard curve drawing.Take an enzyme label plate and add the reagent according to the following table data :(the linear range of this scheme is 2.5-50ug/ml.

     

    Hole no

    A

    B

    C

    D

    E

    F

    G

    H

    Protein standard(ul) 

    0

    0.5

    1

    2

    4

    6

    8

    10

    deionized water(ul)

    20

    19.5

    19

    18

    16

    14

    12

    10

    bradford dyeing liquid(ul)

    200

    200

    200

    200

    200

    200

    200

    200

    protein content(ng)

    0

    50

    100

    200

    400

    600

    800

    1000

    the final volume

    220

     

    Note: it is best to make 6 protein samples of the same concentration for each gradient to reduce the effect of human error on standard curve rendering.

    4. After mixing, place at room temperature for 5-6 minutes.

    5. The optical absorption value at (595-620) nm was measured with an enzyme marker, and the optical absorption value without BSA was taken as a blank control.

    6. Draw the standard curve with protein content (schedule g) as the abscissa and light absorption as the ordinate.

    7. Sample measurement: dilute the protein sample to proper concentration with deionized water. Take 10 liters of sample, add 200 liters of lBradford dye solution and 10 liters of deionized water.

    8. According to the measured absorption value, the protein content of the sample can be found on the standard curve.

    Experimental data:

    (1).Determination of standard curve: R2=99.71% = 99.5%(figure 1)

    (2).Precision test: mean RSD =0.09%<2%

    (3).Stability test: mean RSD =0.92%<3%

    (4).Repeatability test: mean RSD =0.60%<3%

    (5).The average RSD was 2.7936%<5%

    Common problem:

    1. When the absorbance is greater than 0.8a, please dilute the sample before determining.

    2. When determining protein concentration by the Bradford method, there was a good linear relationship between the concentration of the sample in the range of 2.5-50ug/ml.You can also adjust the standard protein dilution concentration range as needed to plot the standard curve for more accurate results.

    Use plastic or glass cuvettes best.If you use a quartz colouring dish, wash immediately with a small amount of 95% ethanol to wash off the dye after repeated cleaning.Plastic colouring should never be soaked in ethanol or acetone for long periods of time.

    4. The colorimetric sequence starts from the tube with low protein concentration, with no need to clean the colorimetric dish in the middle, so as not to affect the result.

    5. Hydrophobic protein or mucin may precipitate with the dye, and the addition of 1M NaOH can be eliminated.

    6. If the general protein concentration of the sample is known, the standard curve can be made by using a similar 3-tube.

    7. Due to the different contents of arginine and aromatic amino acids in various proteins, Bradford method has a large deviation in the determination of different proteins.

    Bradford dyes can be irritating or corrosive. Wear latex gloves when handling.If the skin, eyes, or with a large amount of water or saline rinse.

    9. The coomassie brilliant blue in the Bradford dye solution is easy to gather into a group. Please turn the bottle upside down for 6 to 8 times before each use and hold the bottle head vertically for horizontal circle action.

    10. Low temperature will reduce the sensitivity of Bradford stain solution. Therefore, Bradford stain solution should be restored to room temperature before use.

    11. In the preparation of standard curve, if the suction quantity is not accurate or the sample adding gun is not accurate, the correlation coefficient of the standard curve will be reduced. The product can be prepared according to the need of times ratio gradient dilution, or the sample adding gun with high accuracy can be used.

    12. Since the reaction of color does not increase proportionately when the protein concentration in Bradford increases to a certain point, the standard curve obtained can be approximately regarded as a straight line within a certain range. The relatively accurate protein concentration should be calculated each time according to the standard curve actually measured.

    13. An enzyme marker is required, and the optimal detection wavelength is 595nm. It can also be measured between 570-610nm, but it will reduce some sensitivity.And 96 holes are needed.If you do not have an enzyme marker, you can use a regular spectrophotometer.When using a spectrophotometer to determine protein concentration, the number of samples that each kit can determine may be significantly reduced.

    14. Unlike some other protein concentration assays (including the Lowry method), the Bradford method does not affect the concentration of proteins in most samples.The concentration of mercaptoethanol in the sample can be as high as 1M, and the concentration of dimeritol can be as high as 5mM.However, it is affected by slightly higher concentration of detergent.To ensure that SDS is lower than 0.125%,Triton x-100 is lower than 0.125%, and Tween 20 is lower than 0.06%, the effects of interfering substances can be eliminated by means of ultra-pure water dilution, dialysis/desalination, and re-dissolution of ACETONE/TCA precipitated proteins.Samples containing detergent is recommended to use Mbchem ™ BCA protein concentration determination kit (Mbchem ™ M3020).  

     

    Features

    • Model: AI-BTLP01
    • 999 Units in Stock

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    Shipping info

    $50.00 to United States

    Contact us to order

    +1(206)6369998 +1(503)2681752


    This product was added to our catalog on Wednesday 13 February, 2019.