Western blot, 0.25-0.5 µg/ml, Human, Mouse, Rat Immunohistochemistry (Paraffin-embedded Section), 2-5 µg/ml, Human Immunocytochemistry/Immunofluorescence, 5 µg/ml, Human Immunofluorescence, 5 µg/ml, Human Flow Cytometry (Fixed), 1-3 µg/1x10 6 cells, Human
Flow Cytometry analysis of JK cells using anti-AGL antibody. Overlay histogram showing JK cells (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-AGL Antibody (1 µg/1x10 6 cells) for 30 min at 20C. DyLight488 conjugated goat anti-rabbit IgG (5-10 µg/1x10 6 cells) was used as secondary antibody for 30 minutes at 20C. Isotype control antibody (Green line) was rabbit IgG (1 µg/1x10 6) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.
WB analysis of AGL using anti-AGL antibody.Lane 1:human Jurkat cell,2:h
IF analysis of ABI1 using anti-ABI1 antibody. ABI1 was detected in a paraffin-embedded section of human cervical cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 5 µg/mL rabbit anti-ABI1 Antibody overnight at 4C. DyLight488 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.
IF analysis of AGL using anti-AGL antibody and anti-Beta Tubulin antibody. AGL was detected in immunocytochemical section of U2OS cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 µg/mL rabbit anti-AGL Antibody and mouse anti-Beta Tubulin antibody overnight at 4C. Cy3 Conjugated Goat Anti-Rabbit IgG and DyLight488 Conjugated Goat Anti-Mouse IgG were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.
IHC analysis of AGL using anti-AGL antibody. AGL was detected in a paraffin-embedded section of human placenta tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 µg/ml rabbit anti-AGL Antibody overnight at 4C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit with DAB as the chromogen.
Western blot analysis of AGL using anti-AGL antibody. Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human Jurkat whole cell lysates, Lane 2: human RT4 whole cell lysates, Lane 3: human K562 whole cell lysates, Lane 4: rat heart tissue lysates, Lane 5: rat liver tissue lysates, Lane 6: mouse heart tissue lysates, Lane 7: mouse liver tissue lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-AGL antigen affinity purified polyclonal antibody at 0.5 µg/mL overnight at 4C, then washed with TBS-0.1% Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit with Tanon 5200 system. A specific band was detected for AGL at approximately 175 kDa. The expected band size for AGL is at 175 kDa.
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