Bradford dye absorption spectrum question?

In summary, the best wavelength to measure protein concentration with Bradford dye is 595 nm due to the wide separation of peaks between acidic and non-acidic forms of the dye, and the ability to use absorption measurements to take ratios for a more accurate measurement. However, caution should be taken when using a "modified" assay. If this is a homework assignment, it should be posted in the appropriate Homework Forum.
  • #1
fightboy
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https://www.physicsforums.com/attachments/74483 https://www.physicsforums.com/attachments/74483
Based in the absorption spectra above, why is 595 nm the best wavelength to measure protein concentration with Bradford dye (Hint: there are two important reasons for choosing 595).
I think it has something to do with the color wheel but honestly the explanation in the manual is very unclear. Can someone try to explain it to me?
 

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  • #2
fightboy said:
https://www.physicsforums.com/attachments/74483 https://www.physicsforums.com/attachments/74483
Based in the absorption spectra above, why is 595 nm the best wavelength to measure protein concentration with Bradford dye (Hint: there are two important reasons for choosing 595).
I think it has something to do with the color wheel but honestly the explanation in the manual is very unclear. Can someone try to explain it to me?

You are asking a question but provide a hint. Is this homework?

You had to make me read up on the local roolz: "Helpers: don't provide the entire solution". [ https://www.physicsforums.com/threads/guidelines-for-students-and-helpers.686781/ ]

I haven't done anything like that, so mind that maybe I'm not helping at all because I could be misunderstanding the procedure.

FWIW, but after looking around the web I'm guessing the wide separation of peaks between acidic and non-acidic forms of the dye is part of one of the two reasons. The other reason could be related to that, by the quantitative use of the absorption measurements, taking ratios (which may or may not be called a "modified" assay - here is where I would urge caution based on my unfamiliarity with the subject).

Now why would I make those two guesses?
 
  • #3
fightboy said:
Based in the absorption spectra above, why is 595 nm the best wavelength to measure protein concentration with Bradford dye (Hint: there are two important reasons for choosing 595).

It's pretty clear from your spectra. Echoing Torbjorn, why are you providing 'hints'?
 
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I have an unquenchable thirst for chemical research over the best or optimal wavelength to measure the blue color represented as a global maximum. :DD
 
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  • #5
In case this is homework:

Since I posted my previous comment, I have noticed that (not attempted) homework has another place.

"Homework Guidelines
  • Posts Belong in the Homework Forum
Any and all high school and undergraduate homework assignments or textbook style exercises for which you are seeking assistance are to be posted in the appropriate forum in our Homework & Coursework Questions area--not in blogs, visitor messages, PMs, or the main technical forums. This should be done whether the problem is part of one's assigned coursework or just independent study. The reason for this is that the scientific and mathematical sections of Physics Forums are to be reserved for discussions and not academic assistance. Since graduate level assignments are meant to be more thought provoking (and hence more worthy of discussion), graduate level questions will be allowed in the relevant part of the main section of PF, provided that the graduate student attempts the problem and shows his work."

[ https://www.physicsforums.com/threads/physics-forums-global-guidelines.414380/ ]
 

1. What is the Bradford dye absorption spectrum?

The Bradford dye absorption spectrum is a graphical representation of the absorption of light by the dye Coomassie Brilliant Blue G-250. This dye is commonly used in protein assays to measure the concentration of proteins in a solution. The spectrum shows the wavelengths of light that are absorbed by the dye, which can then be used to determine the amount of protein present in a sample.

2. How is the Bradford dye absorption spectrum measured?

The Bradford dye absorption spectrum is measured using a spectrophotometer, which is a scientific instrument that can measure the intensity of light at different wavelengths. A solution containing the Bradford dye and a protein sample is placed in the spectrophotometer, and the absorbance of light is recorded at different wavelengths. The resulting data is then plotted to create the spectrum.

3. What does the shape of the Bradford dye absorption spectrum indicate?

The shape of the Bradford dye absorption spectrum indicates the different wavelengths of light that are absorbed by the dye. The peak of the spectrum, where the most light is absorbed, is used to determine the concentration of protein in a sample. The shape of the spectrum can also provide information about the purity of the protein sample and the presence of any contaminants.

4. What factors can affect the Bradford dye absorption spectrum?

The Bradford dye absorption spectrum can be affected by several factors, including the concentration and purity of the protein sample, the concentration of the Bradford dye, and the pH of the solution. Contaminants in the sample can also affect the spectrum, as well as the type and quality of the spectrophotometer used to measure it.

5. How is the Bradford dye absorption spectrum used in protein assays?

The Bradford dye absorption spectrum is used in protein assays to determine the concentration of proteins in a sample. This is done by comparing the absorbance of the protein sample at the peak of the spectrum to a standard curve. The standard curve is created by measuring the absorbance of known concentrations of protein and plotting them on a graph. The concentration of the protein sample can then be determined by finding where its absorbance falls on the standard curve.

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