User Manual V1.0.2
34
We bring light to your sample!
Photopette
®
NOTE:
It is important to check for the pH of a sample measured on our instrument as acidic and
basic solutions will vary the absorbance value.
Direct Protein
In the iOS application, under the Measurement Type, select the Direct Protein.
Total protein concentration can be determined by the measurement of absorbance at 280 nm. This
is a simple and quick way of quantification and the protein sample is recoverable. Proteins
absorb light at 280 nm, primarily the aromatic amino acids (tryptophan and tyrosine), and the
disulfide bonds (S-S bonds) in the protein sequence. The amount of light absorbs by the protein
is dependable on the chemical composition of the protein
–
the number and type of amino acids.
Protein absorption is also affected by the protein structure. Therefore, conditions such as
temperature, pH ionic strength, and presence of detergents can affect the absorption of the proteins.
Calculations
To compute the concentration of a purified protein solution, the formulas are given as:
1.
A
260
/A
280
Ratio: To check for a pure protein, the A
260
/A
280
Ratio is approximately about
0.58.
2.
To eliminate the presence of nucleic acids in the protein sample, the protein concentration
can be estimated according to Warburg and Christian formula:
Concentration (mg/ml) = (1.55 x A
280
)
–
(0.76 x A
260
)
3.
For better calculation of proteins:
Protein concentration (mg protein or peptide per ml) = (A280 x DF x MW) /
ε
Where A
280
is the absorbance of the solution at 280 nm in a 1-cm cell
DF is the dilution factor
MW is the molecular weight of the protein/peptide
ε
is the molar extinction coefficient of each chromophore at 280 nm
