ELISA beginner - bacterial protein (Aug/26/2005 )
I would like to perform a sandwich ELISA to look at the expression of a bacterial protein expression over time as the cells grow through lag, log, and stationary phase. I am taking aliquots of my cells at different ODs and pelleting and freezing the samples. My first problem is that I have no idea how many cells to run in the assay (I have a good antibody and the protein should be well expressed). My second problem is that I want to add the same amount of cells to the assay from the various time points and I don't know how to equalize them. Has any one done similar types of ELISAs?
ELISA's can be a great source of data if can optimise them and get them working on a regular basis, but they usually take a while to optimise which can be frustrating.
Firstly, you need to think about your coating procedure, this involves selecting the right kind of plate/plastic chemistry (try Corning or Nunc websites for advice) and then selecting an amount of antigen that will saturate the available surface area within the well. If you are running ELISA assays using whole bacterial cells (or even purified protein) you should quantify the protein in your samples so that you can be acurate about how much you use in your assay. Sigma provide a very good protein quantification (Modified Lowry Assay) which can be used to quantify protein in intact bacterial cultures or samples, this is a very easy assay and can be done in 4 hours-ish. Use this kit to quantify and then you can titrate your coating antigen out and see if this makes any difference to the ODs you get from your assay. Ultimately, this should allow you to see the saturation point of your coating procedure, this also saves quite a bit of your precious antigen!
Hope this helps...
I don't know if this is still helpful. (eg timely) but if I were you, I would do a western analysis on the crude cell lysates rather than an ELISA,especially since you have a good antibody. IF on the other hand, you planned to semi-purify the time points, then ignore what I am about to say below.
We do this for our bacterial proteins, at different time points.
here's an example:
before IPTG - take 1.0 mL (cells are at 0.5 OD)
1 hr, 3hr, 6 hr or o/n, take 0.5 mL of cells (I grow at 16 C, so by o/n, the OD is only 1.5 or 2)
Spin all of the down,
resuspend in 0.5 mL PBS + 0.5 mL 2x SDS-PAGE sample buffer
Boil 95C for 5-15min.
Add reducing agent (100mM DTT final or 2.5% Beta-mercaptoethanol)
if the lysate is very sticky, you can briefly sonicate for a few secs.
Run ~20 uL on a gel (usually I do a gradient gel)
Transfer and blot
Seems to take more work. But I think your bacterial lysates are going to give you high ELISA backgrounds.