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I ordered 3 antibodies from Abcam on 1/3/12, and 2 of them worked quite well for western blot. However, Rb-anti-Fryl (ab95065) did not work in my western blots and I am wondering if you can provide troubleshooting suggestions or a refund.
This antibody is supposed to recognize a band at 340 kDa and has been shown to detect mouse Fryl. My samples are synaptosomes prepared from mouse cortex and lysed for western blot. I used this antibody at 1:2,000 in either 3% BSA + azide or 1% milk. I also tried boiling the lysate or only heating it to 37C. I am attaching the results I obtained from these western blots (I tried 0.2 ug, 1 ug, and 5 ug per lane).
Asked on Feb 01 2012
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.
Answered on Feb 01 2012