Dear users,

I'm now struggling for almost three months to make a pSTAT3-ELISA (https://www.rndsystems.com/products/human-mouse-phospho-stat3-y705-duoset-ic-elisa_dyc4607b-2) work but was unable to succeed. I completely followed their protocol without the smallest deviation but got no result. I then tried different Lysis Buffers (RIPA with SDS, RIPA with Triton-X-100, Lysis Buffer #6 of RnD) but nothing changed. I doubled the antibody amount with no effect and tried different ug amounts of the samples (1-2,5-5-10-25-50-100ug) but nothing changed. It's always the same result since the beginning: The standard curves are perfect but no signal from the samples. When I use other techniques with the same samples (Western Blot, Dot Blot) I get very good results for pSTAT3. I even performed a Western Blot with the Standards which indicated that my samples should be in the standard range. What can I do to make this ELISA work? Thank you very much for your answers.

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