Anti-Parkin antibody [Prk 8] - BSA and Azide free (ab22259)

Thanks a lot for your trouble-shooting.

As to the lysis buffer, I did not use the RIPA buffer; I used Reagent A from Pierce kit Product # 89874, which was used for cells before doing Dounce Grinder strokes. They did not release the component of Reagent A.

I have one question about the storage condition of this antibody.

You recommended aliquotting and storing at -80°C (add 1% BSA for extra stability), instead of 4oC.

How can I add 1% BSA in? Just weight 1mg BSA and add it into the 100ul-tube?

Thanks!

ANSWER:

Tanya is unfortunately still not in today and I would like to reply on her behalf and try to help you to prevent further delays.

Tanya raised the point of the lysis buffer as it may be that it is not suitable for the extraction of parkin. I have found an extraction procedure from a paper (not using our antibody) which works well at extracting the protein: "Mouse tissues were homogenized at 4 °C either in lysis buffer 1 (0.32 sucrose, complete TM Protease inhibitor, Roche) for ß-actin, parkin and calcium/calmodulin-dependent serine protein kinase (CASK) or in Triple-Detergent-Lysis (TDL) buffer (50 mM HEPES, 150 mM NaCl, 25 mM EDTA, 1% Nonidet P40, 0.5% Na-deoxycholate, 0.1% SDS and protease inhibitor cocktail tablets (Roche) for ß-actin and synphilin-1 Western blot. Samples were chilled on ice for 30 min and centrifuged at 14.000 rpm in a precooled centrifuge. Supernatant was diluted 1:1 in modified 2× Laemmli sample buffer (125 mM Tris–HCl pH 6.8, 6% SDS, 10% ß-mercaptoethanol, 30% glycerin) and boiled for 5 min at 100 °C." Parkin expression in the developing mouse.Kuhn K, Zhu XR, Lubbert H, Stichel CC. Brain Res Dev Brain Res. 2004 Apr 19;149(2):131-42.

Tanya also suggested running a positive control along your MES samples and I also agree that this will help you be sure the levels of parkin are not too low in your samples. As you can see in the paper referenced below levels of parkin are very low in the embryo, and become higher during development, reaching a maximum at adulthood, so I would recommend to run adult mouse brain lysate.

The datasheet is a little confusing I have to say regarding the best storage conditions for this antibody. we recommend to aliquot the antibody (neat) at store at -20/-80 OR if you want it diluted to add a little BSA up to a dilution of the antibody to 50ug/ml. But it is best to keep it undiluted and stored in aliquots at -20/-80.

I hope this clarification helps and the information above too, please do not hesitate to contact us again if you need further assistance,