Anti-FRYL antibody (ab95065)

I will be happy to help you with this.

Our datasheet (https://www.abcam.com/fryl-antibody-ab95065.html) shows data similar to what your are seeing: many bands at lower molecular weights but also a single band at the expected size. These samples are cell lysates, and I think your samples, from cortex, are going to be more problematic for this antibody, which cross-reacts with other proteins. My guess is that the amount of Fryl in the cell line lysates as a proportion of total protein may make it easier to isolate a Fryl signal in blots of these lysates than in blots of your samples, if Fryl is present in the synaptosomes as a smaller proportion of total protein than in these cell lines.

Farther down the datasheet , another blot of HeLa lysates suggests that it is necessary to IP the protein in order to see a clean Fryl signal. But it is not clear to me that your blots show any signal for Fryl, so I cannot say IP will help you.

The laboratory that validated the antibody for western blotting used HiMark molecular weight markers and NuPage 3-8% Tris-acetate gels (cat# EA03752BOX) with the recommended buffers (all from Invitrogen).

My advice is to optimize the transfer for large proteins. I recommend removing methanol from the transfer buffer and including SDS at a concentration of 0.1%, to encourage movement of Fryl out of the gel, onto PVDF.

We have one other Fryl antibody, ab95011, raised against a different region, but the WB data looks very similar, with some cross-reactivity at lower molecular weights. I will be happy to send this a free replacement if you want to try it, or a credit or refund.

I look forward to your reply.