Dear Rony

first of all to perform measurment at 280nm in microplates, you need to use the plates with flat bottom trasparent to UV because otherwise as happen in cuvette if you are using standard plastic instead quarz the UV will be not able to pass through the plastic and you will not measure the absorbance of the sample but of the plate.

Moreover as you probably well know the absorbance is regulated by lambert-beer law

A=ebc where b is the optic path-lenght and while this value is costant and generally = 1 (1cm) in the cuvette, and this happen also if you fill the cuvette with different volumes, when you are using a plate, the optic path-lenght change in function of the volume

eg optic path-lenght is double if you are using 200ul/well respect when you are using 100ul/well

i think that the ratio that you mention, 1:3 is whats happen when you are using 200ul, however i suggest to you just to perform a calibration with BSA in the cuvette, and afterward transfer 100ul and 200ul of the samples from cuvette to plate and repeat the measurement. In this way in one single and simple experiment you will carefully determine that ratio for your specific plates.

good luck

Manuele

Hello

If you have an standard protein with known concentration, make a serial dilution and draw an standard curve. In the same micro-plate you can have your unknown samples too. We usually do not consider a coefficient value separately.

Good luck

First of all, as per my experience A280 may not be a really good method to estimate proteins because it provides an estimation of only aromatic proteins. It is true that all methods do have some or the other biases. But, I would recommend BCA method or Bradford for estimation. Both these methods, like any other are not perfect, and do give overestimations. But the idea is if they do give some overestimation, they would do so for all samples examined. So it would be relative. Anyway, plot a standard curve using known concentrations of BSA and estimate your protein of choice. But the standard curve using BSA would not truly represent your sample in case of A280 measurement, because of the enormously varied expression of amino acids in different samples.