I've come across a neat protocol for measuring immune response in plasma which uses bioluminescence. However, I was wondering if it was possible if the same samples can then be processed for western blot. Thanks in advance.
Hi David, in principle you can do it, but you have to keep an eye on a few things.
First, as always when you work with non-denatured proteins work in cold room or on ice to prevent degradation.
Secondly, check the protein concentration. On WB you will need at least 5-10 ug of protein per lane (but you may need more, this will depend on the relative abundance of your protein in the extract and the affinity of the antibody, you can measure this by loading a serial dilution of your extract starting from 40 ug and diluting by a two-fold factor until 5 ug, or less if you wish!), which means - in these convenient conditions - having a concentration of at least 0.2-0.5 ug/ul (considering you can load 20-25 ul in a well). Bioluminescence reactions are very sensitive, will probably work with less concentration and larger volumes...
If your protein concentration is too low, you can still concentrate. There are several possibilities, Centricon columns (Millipore) or precipitation (10 vol of acetone at -80°C, acetone/methanol mixes, etc... you'll find them on the internet!)
In any case you'll have to add some Laemmli buffer or similar and boil your proteins before loading on gel. Normally you have these buffers at a 5x concentration, so you'll have to consider a 20% dilution of your protein extracts to be able to load them on gel.