Anti-AKR1D1 Antibody (monoclonal, 6I4)

• Catalog Number: orb654286
• Category: Antibodies
• Description: Anti-AKR1D1 Antibody (monoclonal, 6I4). Tested in Flow Cytometry, IHC, WB applications. This antibody reacts with Human, Mouse, Rat.
• Species/Host: Mouse
• Clonality: Monoclonal
• Clone Number: 6I4
• Tested applications: FC, IHC, WB
• Reactivity: Human, Mouse, Rat
• Isotype: Mouse IgG2b
• Immunogen: A synthetic peptide corresponding to a sequence at the C-terminus of human AKR1D1, which shares 90.9% and 93.9% amino acid (aa) sequence identity with mouse and rat AKR1D1, respectively.
• Antibody Type: Primary Antibody
• Concentration: Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Form/Appearance: Lyophilized
• Conjugation: Unconjugated
• MW: 37 kDa
• UniProt ID: P51857
• Storage: Maintain refrigerated at 2-8°C for up to 2 weeks. For long term storage store at -20°C in small aliquots to prevent freeze-thaw cycles.
• Alternative names: 3-oxo-5-beta-steroid 4-dehydrogenase; Aldo-keto re
• Note: For research use only
• Application notes: Western blot, 0.1-0.5μg/ml, Human, Mouse, Rat Immunohistochemistry (Paraffin-embedded Section), 0.5-1μg/ml, Human Flow Cytometry (Fixed), 1-3μg/1x106 cells, Human. Add 0.2ml of distilled water will yield a concentration of 500ug/ml
• Expiration Date: 12 months from date of receipt.

Flow Cytometry analysis of HepG2 cells using anti-AKR1D1 antibody. Overlay histogram showing HepG2 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 mouse anti-AKR1D1 Antibody (1 µg/1x106 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (5-10 µg/1x106 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1 µg/1x106) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

IHC analysis of AKR1D1 using anti-AKR1D1 antibody. AKR1D1 was detected in paraffin-embedded section of human liver 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 1 µg/ml mouse anti-AKR1D1 Antibody overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.

IHC analysis of AKR1D1 using anti-AKR1D1 antibody. AKR1D1 was detected in paraffin-embedded section of human liver 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 1 µg/ml mouse anti-AKR1D1 Antibody overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.

Western blot analysis of AKR1D1 using anti-AKR1D1 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 50 ug of sample under reducing conditions. Lane 1: human HepG2 whole cell lysates; Lane 2: human HL-60 whole cell lysates; Lane 3: human THP-1 whole cell lysates; Lane 4: rat liver tissue lysates; Lane 5: rat RH35 whole cell lysates; Lane 6: mouse liver tissue lysates; Lane 7: mouse HEPA1-6 whole cell 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 mouse anti-AKR1D1 antigen affinity purified monoclonal antibody at 0.5 µg/mL overnight at 4°C, then washed with TBS-0.1% Tween 3 times with 5 minutes each and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:10000 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 AKR1D1 at approximately 37 KD. The expected band size for AKR1D1 is at 37 KD.