I am testing insoluble homogenates diluted to 0.25M guanidine HCL. Standards for every kit tested work beautifully (standards also in guanidine HCL dilution. The samples are from human gene KI mice brain homogenates. The only reason I can think of for these samples to not work is that the kit's transcript versus the KIs don't match. Has anyone else had this happen?
One thing you could try is a dot-blot instead of ELISA. Ideally, this is done with a 96-well vacuum manifold (Millipore), but if you can't lay your hands on one, just pipett 1-2 µl of sample onto a PVDF membrane (wetted with methanol, washed in dd water and equilibrated with PBS) and allow to air-dry.
The membrane is then developed just like a western. This method has a number of advantages over ELISA:
- no capture antibody or activating chemistry on the plate are required
- you capture essentially 100% of the protein in your sample (about 60% with nitrocellulose), the binding step with plates captures usually only a few %. As a result, dot blots are 2 orders of magnitude more sensitive than ELISA
- Capture works well even in the presence of caotropes or detergents (not sure about that with NC, I always use PVDF)
- Washing and detection steps are quicker, you don't need a pipetting robot
- PVDF membranes are cheaper than plates
- With chemiluminescence detection you get a linear signal over 5-6 orders of magnitude, absorbance measurement in ELISA gives you only one. For that purpose I use a simple adapter that allows insertion of the membrane in a plate luminometer.
Thank you. Do you cut the spot off the membrane to read on a plate? How does that work? ie...loving the ease of this idea!
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