Protein Concentration Correction

What is your method for determining protein concentration?

We use a corrected Absorbance at 280 nm, and an extinction coefficient (0.1% w/v) of 1.4 to determine antibody concentration.

The scatter correction derivation is shown below:

A280 = A°280 + Isc,280

Where:

A280 = Observed absorbance.

280 =  Actual intrinsic absorbance of the protein.

Isc, 280 = Absorbance due to scatter or dispersion.

A280 =  A°280 + Isc,280

280 = A280 – Isc,280

Isc,280 is proportional to 1/λ4 and equal to  K (1/λ4) = K (1/(280)4

Where K = proportionality constant.

Likewise:

A320 = A°320 + Isc,320

For proteins, A°320 = 0.   (Not valid for proteins absorbing at 320 nm or for proteins larger than λ (e.g. von Willebrand factor).

Therefore:

A320 = Isc,320 = K(1/(320)4)

K = A320 x (320)4 = Isc,280 x (280)4

Isc,280 = A320 ((320)4/(2804) = 1.7 x A320

And:

280 = A280 – 1.7(A320)

[Our regards to Dr. Sriram Krishnaswamy for his helpful comments.]

 

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