Hi everyone,
we have a nanophotometer here that can measure protein concentrations via UV absorbance. However, as usual, one has to define the extinction coefficient for your given protein.
I now would like to measure the protein concentration in cell lysates of Jurkat cells that I obtained. Our ELISA reader is broken and I'm looking for a quick alternative.
Is there an estimation of an extinction coefficient that I could use for this nanophotometer to determine the overall quantitiy of protein in my samples? Would it be possible to use e.g. the coefficient for GAPDH as a representative or would the risk to measure something else which is not normalized in the cells be too high?
Thank you for any input,
best regards,
Gregor
You can not use UV absorbance at 280 nm to measure protein concentration in cell lysates, because there are too many compounds that also absorb light, e.g., DNA at 260 nM.
For measuring protein concentration in cell lysates, you can use assays like Bradford assay, but please keep in mind that there are also many limitations, depending on your lysis buffer. Many detergents that are usually present in cell lysis buffers are not compatible with this assay and have to be removed before the assay. It would be very important to include control samples in your assay to see the effect of buffer ingredients.
Thank you for your answer Andreas Werner, that's what I figured. Bradford seems the way to go, keeping in mind your hints reagarding detergents.
Thank you!
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