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About this calculator
What it does
It takes the absorbance at 280 nm and the absorbance at the dye maximum, each with the dilution it was read at, and puts both on the undiluted sample. It removes the dye's share of the 280 nm reading and reports the protein concentration in molar (and in mg/mL when you enter a molecular weight), the dye concentration and the degree of labelling.
How to use it
Enter the two readings and the dilution of each, pick the dye, enter the protein's extinction coefficient and the path length, and read the result. The page opens on a worked example; the two example buttons fill every field for you, and Clear empties them.
How it works
Relations
- A280, undiluted equivalent = A280 as read × N (that reading's own dilution)
- Dye-maximum absorbance, undiluted equivalent = absorbance at the dye maximum as read × N (that reading's own dilution)
- Dye term = dye-maximum undiluted equivalent × CF; corrected A280 = A280 undiluted equivalent − dye term
- Protein molarity = corrected A280 / (protein extinction coefficient × path length)
- Dye molarity = dye-maximum undiluted equivalent / (dye extinction coefficient × path length)
- Degree of labelling = dye molarity / protein molarity
- Protein mg/mL = protein molarity × molecular weight, when you enter the molecular weight in g/mol
Constants and assumptions
The vendor's instruction, quoted: "". It is shown as a note beside any reading above 2.0 as read; it does not change the numbers.
The free-dye sentence, quoted: "".
Precision, rounding and engine version
The chain is computed exactly, with no rounding between the readings and the printed values. A value is rounded once, when it is printed.
Flags
Why not a spreadsheet?
A spreadsheet leaves the dilution of each reading, the dye lot and the rounding to memory. This calculator asks for each reading's own dilution, keeps full precision until it prints, and shows the working with your numbers.