bradford protein assay


  • By using the Bradford protein assay, one can avoid all of these complications by simply mixing the protein samples with the Coomassie brilliant blue G-250 dye (Bradford reagent)
    and measuring their absorbances at 595 nm, which is in the visible range[8] and may be accuretaly measured by the use of a mobile smartphone camera.

  • This standard curve is then used to determine the concentration of the unknown protein.

  • [21] Using a broad range of protein concentration will make it harder to determine the concentration of the unknown protein.

  • Using data obtained to find concentration of unknown[edit] In summary, in order to find a standard curve, one must use varying concentrations of BSA (Bovine Serum Albumin)[2]
    in order to create a standard curve with concentration plotted on the x-axis and absorbance plotted on the y-axis.

  • To do this, one must divide concentration by volume of protein in order to normalize concentration and multiply by amount diluted to correct for any dilution made in the protein
    before performing the assay.

  • [18] Much of the non-linearity stems from the equilibrium between two different forms of the dye which is perturbed by adding the protein.

  • Actual assay data for determine concentration of unknown based on line of best fit of the above standard curve In order to attain a concentration that makes sense with the
    data, the dilutions, concentrations, and units of the unknown must be normalized (Table 1).

  • This process is more beneficial since it is less pricey than other methods, easy to use, and has high sensitivity of the dye for protein.

  • If nucleic acids are present in the sample, they would also absorb light at 280 nm, skewing the results further.

  • [6] Other interference may come from the buffer used when preparing the protein sample.

  • This will not be a problem if a low concentration of protein (subsequently the buffer) is used.

  • [9] Disadvantages The Bradford assay is linear over a short range, typically from 0 µg/mL to 2000 µg/mL, often making dilutions of a sample necessary before analysis.

  • [15] The linear graph acquired from the assay (absorbance versus protein concentration in μg/mL) can be easily extrapolated to determine the concentration of proteins by using
    the slope of the line.

  • [5] The increase of absorbance at 595 nm is proportional to the amount of bound dye, and thus to the amount (concentration) of protein present in the sample.

  • [1] It is a quick and accurate[2] spectroscopic analytical procedure used to measure the concentration of protein in a solution.

  • [11] However, there are some deterd protein, it is possible that the concentration measured will be inaccurate.

  • Changes to the original method, such as increasing the pH by adding NaOH or adding more dye have been made to correct this variation.

  • Although these modifications result in a less sensitive assay, a modified method becomes sensitive to detergents that can interfere with sample.

  • In a large scale, one must compute the extinction coefficient using the Beer-Lambert Law in which A is the measured absorbance, ε is the slope of the standard curve, L is
    the length of the cuvette, and C is the concentration being determined.

  • [1] During the formation of this complex, the red form of Coomassie dye first donates its free electron to the ionizable groups on the protein, which causes a disruption of
    the protein’s native state, consequently exposing its hydrophobic pockets.

  • When the dye binds to the proteins through a process that takes about 2 minutes, a change in the absorption maximum of the dye from 465 nm to 595 nm in acidic solutions occurs.

  • Only the molecules that bind to the proteins in solution exhibit this change in absorption, which eliminates the concern that unbound molecules of the dye might contribute
    to the experimentally obtained absorption reading.

  • While other detergents interfere with the assay at high concentration, the interference caused by SDS is of two different modes, and each occurs at a different concentration.

  • [6] Unlike other protein assays, the Bradford protein assay is less susceptible to interference by various chemical compounds such as sodium, potassium or even carbohydrates
    like sucrose, that may be present in protein samples.

  • [3] Under acidic conditions, the red form of the dye is converted into its blue form, binding to the protein being assayed.

  • When more than one solution is tested, it is important to make sure every sample is incubated for the same amount of time for accurate comparison.

  • [7] Advantages Many protein-containing solutions have the highest absorption at 280 nm in the spectrophotometer, the UV range.

  • Same test tubes cannot be used since the stain would affect the absorbance reading.

  • Actual BSA data attained from a micro scale UV-Vis Spectrophotometer The equation displayed on the chart gives a means for calculating the absorbance and therefore concentration
    of the unknown samples.

  • The anionic bound form of the dye which is held together by hydrophobic and ionic interactions, has an absorption spectrum maximum historically held to be at 595 nm.


Works Cited

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PMID 942051.
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ISBN 9780470087664. OCLC 420027217.
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PMID 35987416. S2CID 251684735.
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Burcu; Dınçer, Ayşşe; Habib, Ömer; Zıhnıoglu, Figen (2007-08-01). “Comparison of five methods for determination of total plasma protein concentration”. Journal of Biochemical and Biophysical Methods. 70 (5): 709–711. doi:10.1016/j.jbbm.2007.05.009.
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Ninfa, Alexander J; Ballou, David P; Benore, Marilee (2009). Fundamental Laboratory Approaches for Biochemistry and Biotechnology. Wiley. p. 113.
o ^ Rabilloud, Thierry (2018). “Optimization of the cydex blue assay: A one-step colorimetric protein
assay using cyclodextrins and compatible with detergents and reducers”. PLOS ONE. 13 (4): e0195755. Bibcode:2018PLoSO..1395755R. doi:10.1371/journal.pone.0195755. PMC 5895047. PMID 29641569.
o ^ Zor, Tsaffrir; Selinger, Zvi (1996-05-01). “Linearization
of the Bradford Protein Assay Increases Its Sensitivity: Theoretical and Experimental Studies”. Analytical Biochemistry. 236 (2): 302–308. doi:10.1006/abio.1996.0171. PMID 8660509.
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Quantitation”. Protein Protocols Handbook, the. pp. 15–22. doi:10.1385/1-59259-169-8:15. ISBN 1-59259-169-8.
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