Recombinant Anti-GBA (mutated E326K) antibody [EPR24900-273] (ab302607)

Blocking / Diluting buffer and concentration: 5% NFDM/TBST

Negative in GBA (WT) transfected cells and GBA (WT)-expressing tissues.

Immunohistochemical analysis of paraffin-embedded HEK-293T cells transfected with a human GBA E365K expression vector containing a myc-His tag (Cell Pellet) (A) labeling GBA (mutated E326K) with ab302607 at 1/5000 dilution (0.094 µg/mL), and HEK-293T cells transfected with a human GBA expression vector containing a myc-His tag (Cell Pellet) (B), and HEK-293T transfected with an empty vector (Cell Pellet) (C) followed by a ready to use Leica DS9800 (Bond™ Polymer Refine Detection kit). Cytoplasmic staining on HEK-293T cells observed (Image A). No staining on HEK-293T cells transfected with a human WT GBA expression vector containing a myc-His tag cell pallet (image B) and 293T transfected with an empty vector cell pallet (image C). The section was incubated with ab302607 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Primary diluent was used instead of primary antibody, followed by a ready to use secondary antibody Leica DS9800 (Bond™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue for labeling GBA (mutated E326K) with ab302607 at 1/5000 dilution (0.094 µg/mL) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection kit). Negative control: No staining on human breast. The section was incubated with ab302607 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293 (Human epithelial cells from embryonic kidney) cells transfected with a human GBA E365K expression vector containing a Myc tag and HEK-293T cells transfected with a human GBA expression vector. Confocal image showing cytoplasmic staining in HEK-293T cells transfected with a human GBA E365K expression vector and no staining in HEK-293T cells transfected with only a human GBA expression vector. ab302607 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150081) secondary antibody at 1/1000 dilution (green). The nuclear counter stain is DAPI (blue).

Counterstain antibody was a anti-Myc-Tag Mouse mAb (Alexa Fluor^® 647) (Red).

Secondary antibody only control: primary diluent was used instead of primary antibody followed by preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

Flow cytometric scatter graph showing 4% paraformaldehyde fixed and 90% methanol permeabilized HEK-293T (human embryonic kidney) cells transfected with a human GBA (WT) expression vector containing a myc-his tag (Left) and HEK-293T transfected with a human GBA (mutated E326K) expression vector containing a myc-his tag labeling GBA (mutated E326K) (Right). ab302607 at 1/500 dilution (0.1μl).  Secondary antibody was Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) used at 1/2000 dilution.

GBA (mutated E326K) was immunoprecipitated from 10μg of HEK-293T transfected with a human GBA (E326K mutation) expression vector containing a His-tag whole cell lysate, with ab302607 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab302607 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used as secondary antibody at 1/5000 dilution.

Lane 1: HEK-293T transfected with a human GBA (E326K mutation) expression vector containing a His-tag, whole cell lysate (Input).

Lane 2: ab302607 IP in HEK-293T transfected with a human GBA (E326K mutation) expression vector containing a His-tag, whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab302607 in HEK-293T transfected with a human GBA (E326K mutation) expression vector containing a His-tag, whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 49 seconds.

Observed MW(KDa): 60

Dot blot analysis of GBA (mutated E326K) with ab302607 at a dilution of 1/1000 (0.472 μg/ml). ab97051 (Peroxidase conjugated goat anti-rabbit IgG (H+L)) was used as the secondary antibody at a dilution of 1/100000.

Lane 1: Human GBA peptide a containing E326K mutation

Lane 2:  Lane 2: Human GBA peptide b containing E326K mutation

Lane 3:  Lane 3: Human WT GBA peptide corresponding to the E326K-mutated peptide

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.