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1) Are you still using rat hippocampus lysates? How much protein was loaded per lane of the gel?
Yes , I am and loaded 30ug per lane.
2) Could you describe the electrophoresis and gel conditions (% of the gel etc.)?
I used a 6% tris gel.
3) Could you describe the transfer and blocking conditions (buffer, transfer time, blocking agent etc.)?
I transferred overnight at 4 degrees using sodium bicarbonate, and blocked for 1hr at room temperature.
4) Does the secondary antibody work with other primaries?
Yes other people in the lab are currently obtaining results with this same secondary.
5) Detection method (ECL, ECLPlus etc.)
ECL (pico and femto) on film developer (1hr exposure only displayed background)
6) Did the ladder proteins transfer, or did you confirm transfer of the high molecular proteins via Ponceau stain?
Yes the ladder was transferred properly and confirmed using Ponceau that protein transferred.
Asked on Nov 20 2012
Thank you for sending the additional information and for your patience.
I forwarded your protocol details to the lab for a second look, and we would have expected the antibody to work in the conditions that you've described. I am very sorry that neither of these antibodies has worked for you, and I can arrange a credit or a refund for your original purchase if you'd like. Alternatively, we do have one other antibody that is predicted to work with rat samples, ab135457-
This antibody hasn't been tested with rat samples, but since the immunogen is homologous to the rat protein it may work. I'd be happy to send this out if you'd like to try it.
I apologize again for the poor results with these Nav1.6 antibodies, but if there is anything else that we can do for you, please let me know.
Answered on Nov 20 2012