Product Review: GBA antibody – C-terminal region (ARP61322_P050) in rat organs, fibroblasts and HeLa cells using Western Blot.

Product Page Link: GBA antibody – C-terminal region (ARP61322_P050)

Data provided by Dr. Andrea Balreira

Protocol: SDS-PAGE and WB

Cell culture samples (fibroblasts and HeLa cells) were lysed and rat organs were homogenized, and proteins were quantified by the Bradford technique. 100ug of each sample were subjected to SDS-PAGE and electroblotted onto a nitrocellulose membrane. Membranes were blocked with %5 skim milk and 0.1% Tween-20 in Tris-buffered saline (TBS) for 1h at room temperature and incubated overnight with the anti-GBA antibody (1:1000) at 4C. Membranes were then washed twice with 0.1% Tween-20 in TBS and incubated with the appropriate secondary antibodies (anti-rabbit IgG-alkaline phosphatase antibody (Sigma)) for 1h at room temperature. After washing, detection was performed using alkaline-phosphatase substrates.

PNGase treatment: Samples of fibroblasts lysastes containing 20ug of total protein were treated overnight with PNGase F according to manufacturer’s instruction.

Results:

As can be observed in the WB, anti-GBA antibody was capable of detecting all of the human glucocerebrosidase glycosylated forms. In what concerns rat organs, it seems that it only works in kidney homogenate. It is not clear if rat GBA has the same glycoslation pattern as the human so we are unable to infer the PNGase result.

Note: Compare this study with results in Balreira, A, HMG, 2008 and recombinant forms of this enzyme. The specificity of the antibody was further confirmed by the PNGase assay, that shows us the glycosylated form of GBA (PNGase-) and the PNGase digested form (PNGase +).

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