Anti-AGL/Alpha-glucosidase antibody [EPR8880]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-AGL/Alpha-glucosidase antibody [EPR8880] (AB133720)

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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-AGL/Alpha-glucosidase antibody [EPR8880] (AB133720)

Intracellular Flow Cytometry analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labeling AGL/Alpha-glucosidase with purified ab133720 at 1/80 dilution (10μg/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).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AGL/Alpha-glucosidase antibody [EPR8880] (AB133720)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AGL/Alpha-glucosidase antibody [EPR8880] (AB133720)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AGL/Alpha-glucosidase antibody [EPR8880] (AB133720)

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-AGL/Alpha-glucosidase antibody [EPR8880] (AB133720)

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.

• WB

Lab

Western blot - Anti-AGL/Alpha-glucosidase antibody [EPR8880] (AB133720)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-AGL/Alpha-glucosidase antibody [EPR8880] (ab133720) at 1/5000 dilution

Lane 1:

Human fetal muscle lysates at 20 µg

Lane 2:

Human fetal heart lysates at 20 µg

Lane 3:

Mouse muscle lysates at 20 µg

Lane 4:

Rat muscle lysates at 20 µg

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

false

• WB

Lab

Western blot - Anti-AGL/Alpha-glucosidase antibody [EPR8880] (AB133720)

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:

Western blot - Anti-AGL/Alpha-glucosidase antibody [EPR8880] (ab133720)

Predicted band size: 175 kDa

Observed band size: 170 kDa

false

• WB

Lab

Western blot - Anti-AGL/Alpha-glucosidase antibody [EPR8880] (AB133720)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-AGL/Alpha-glucosidase antibody [EPR8880] (ab133720) at 1/1000 dilution

Lane 1:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates at 20 µg

Lane 2:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates at 20 µg

Secondary

All lanes:

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

Predicted band size: 175 kDa

false

• WB

Unknown

Western blot - Anti-AGL/Alpha-glucosidase antibody [EPR8880] (AB133720)

All lanes:

Western blot - Anti-AGL/Alpha-glucosidase antibody [EPR8880] (ab133720) at 1/1000 dilution

Lane 1:

Human fetal muscle lysate at 10 µg

Lane 2:

Mouse muscle lysate at 10 µg

Lane 3:

Human fetal heart lysate at 10 µg

Lane 4:

K562 cell lysate at 10 µg

Lane 5:

293T cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 175 kDa

false

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-AGL/Alpha-glucosidase antibody [EPR8880] (AB133720)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.