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The technician simply told me that Abcam is not their source for their ApoB standard and that the kit is polyclonal to ApoB 48 and 100, so should be able to detect the ApoB 100. She asked how I handled and stored the protein (to which I responded: dilution followed by aliquoting and storing at -20C), but I have not heard back from her since.
We are not particularly wedded to the use of the (other company's) kit, but have at least 2 more 96-well plates to use before deciding to move on to another company. Do you have any suggestions as to what we could possibly try in order to get better results? Are there any treatments of the protein (apart from repeated freeze/thaw) that we should avoid?
Asked on Jan 26 2012
I am a little concerned about the formulation of ab77859. It is in 200mM Tris, 10mM Sodium decyl sulfate, pH 8.25, and I am wondering if the sodium decyl sulfate concentration is sufficient to denature the protein, possibly making it incompatible with the kit, which I assume has a standard that is not denatured.
The protein has only been validated here in western blotting. If someone in your lab routinely runs SDS-PAGE gels, they might be able to check if the protein is degraded with a Coomassie stain of the gel. It should look something like the blot we show on the datasheet, without much smearing in the lane, which would suggest degradation and a defective product, at least that vial of product.
In any case, I am not sure what to suggest to make this work in your assay. If the kit is working with the kit standards, then either ab77859 is inappropriate for this ELISA, owing to how it is processed, or it is degraded.
Answered on Jan 26 2012