Recently, I am doing preparative SEC for the purification of a protein.
I found that the A280 value usually spikes at a very high value when the elution volume is close to 1 column volume. typically, we consider the A280 relative to protein concentration.
but when I do BCA assay, there is no a spike signal for that.
My current thought is that it was caused by the absorbance variation between PBS ( which is the sample solution) and elution buffer(containing PIPES, EDTA and NaCl)
How you guys think about this?
At one column volume (slightly earlier, actually), all the small molecules that can readily enter the resin beads elute. If SEC is the first step in the purification, every small molecule in the extract will be found there. Most proteins, except the smallest ones, will have eluted earlier. So this late fraction is a mixture of numerous, mostly non-protein substances in the extract. Some of them will absorb in the UV.
Even if SEC is the 2nd step in the purification, some of these UV absorbing small molecules may have carried over from the first step.
I don't think PIPES buffer has significant absorbance at 280 nm.
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