Flow Cytometry analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labeling AGL/Alpha-glucosidase with purified ab133720 at 1:80 dilution (10 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling AGL/Alpha-glucosidase with Purified ab133720 at 1:100 dilution. Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200. ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human skeletal muscle tissue sections labeling AGL/Alpha-glucosidase with Purified ab133720 at 1:2000 dilution (0.4 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP)
secondary antibody was used at 1:500 dilution. PBS instead of the primary antibody was used as the negative control.

All lanes : Anti-AGL/Alpha-glucosidase antibody [EPR8880] (ab133720) at 1/1000 dilution (purified)

Lane 1 : K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates
Lane 2 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

Predicted band size: 175 kDa

Blocking and diluting buffer: 5% NFDM/TBST

All lanes : Anti-AGL/Alpha-glucosidase antibody [EPR8880] (ab133720) at 1/5000 dilution (purified)

Lane 1 : Human fetal muscle lysates
Lane 2 : Human fetal heart lysates
Lane 3 : Mouse muscle lysates
Lane 4 : Rat muscle lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

Predicted band size: 175 kDa
Observed band size: 175 kDa

Blocking and diluting buffer: 5% NFDM/TBST

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: AGL/Alpha-glucosidase knockout HAP1 whole cell lysate (20 µg)
Lane 3: HepG2 whole cell lysate (20 µg)
Lane 4: Hek293 whole cell lysate (20 µg
Lanes 1 - 4: Merged signal (red and green). Green - ab133720 observed at 170 kDa. Red - loading control, ab18058, observed at 130 kDa.

Unpurified ab133720 was shown to specifically react with AGL/Alpha-glucosidase when AGL/Alpha-glucosidase knockout samples were used. Wild-type and AGL/Alpha-glucosidase knockout samples were subjected to SDS-PAGE.
Unpurified Ab133720 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-AGL/Alpha-glucosidase antibody [EPR8880] (ab133720) at 1/1000 dilution (unpurified)

Lane 1 : Human fetal muscle lysate
Lane 2 : Mouse muscle lysate
Lane 3 : Human fetal heart lysate
Lane 4 : K562 cell lysate
Lane 5 : 293T cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 175 kDa

Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) labeling AGL/Alpha-glucosidase with purified ab133720 at 1/250 dilution. Cells were fixed with 4% PFA and permeabilized with 0.1% tritonX-100. ab150077 Goat anti rabbit IgG (Alexa Fluor^®488) at 1/1000 was used as the secondary antibody. Nuclei were counterstained with DAPI. PBS was used instead of the primary antibody as the negative control.

Immunohistochemical analysis of paraffin-embedded Human liver tissue labelled with unpurified ab133720 at 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human muscle tissue labelled with unpurified ab133720 at 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Equilibrium disassociation constant (K_D)
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