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☑ Relative expression (RE)
Western blot analysis of ALDH1A3 on different lysates with Rabbit anti-ALDH1A3 antibody (HA724405) at 1/5,000 dilution.
Lane 1: U-87 MG (Human glioblastoma cells) cell lysate
Lane 2: A431 (Human epidermoid carcinoma skin squamous cells) cell lysate
Lane 3: MCF7 (Human breast cancer cells) cell lysate
Lane 4: 293T (Human embryonic kidney cells) cell lysate
Lane 5: Caco-2 (Human colorectal adenocarcinoma cells) cell lysate
Lane 6: HeLa (Human cervical adenocarcinoma cells) cell lysate
Lysates/proteins at 10 µg/Lane.
Exposure time: 30 seconds; ECL: K1801
Low expression: MCF7 cells (PMID: 31197235).
Low expression of ALDH1A3 protein in 293T, Caco-2, and HeLa cells is consistent with the predicted expression pattern.
Blocking: 5% NFDM/TBST, 1 hour at room temperature
Primary antibody: HA724405, 1/5,000 in primary antibody dilution buffer (K1803), overnight at 4 ℃
Secondary antibody: Goat anti-Rabbit IgG-HRP (HA1001), 1/50,000 in 5% NFDM/TBST, 1 hour at room temperature
Predicted band size: 56 kDa
Observed band size: 50-56 kDa
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Western blot analysis of ALDH1A3 on different lysates with Rabbit anti-ALDH1A3 antibody (HA724405) at 1/5,000 dilution.
Lane 1: Human ALDH1A3 recombinant protein
Lane 2: Human ALDH1A2 recombinant protein
Lane 3: Human ALDH1A1 recombinant protein
Lysates/proteins at 20 ng/Lane.
Exposure time: 7 seconds; ECL: K1801
Blocking: 5% NFDM/TBST, 1 hour at room temperature
Primary antibody: HA724405, 1/5,000 in primary antibody dilution buffer (K1803), overnight at 4 °C
Secondary antibody: Goat anti-Rabbit IgG-HRP (HA1001), 1/50,000 in 5% NFDM/TBST, 1 hour at room temperature
Predicted MW: 56 kDa
Observed MW: 58-61 kDa
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☑ Relative expression (RE)
Application: Immunocytochemistry (IF-cell)
Species: Human
Sample: U-87 MG (Human glioblastoma cells)/MCF7 (Human breast cancer cells)
Fixation: 4% Paraformaldehyde, 15 minutes at room temperature.
Permeabilization: 0.1% Triton X-100, 15 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 1 hour at room temperature.
Antibody dilution buffer: 1% BSA in PBST.
Primary antibody: HA724405, 1/100, overnight at 4°C.
Secondary antibody: Goat Anti-Rabbit IgG (iFluor™ 488, HA1121), 45 minutes at room temperature.
Counterstain: Beta tubulin (HA601187, red), 1/100, overnight at 4℃. The Nuclear counterstain was DAPI (Blue).
Low expression: MCF7 cell (PMID: 31197235).
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☑ Relative expression (RE)
Application: Flow Cytometry (Intra)
Species: Human
Sample: U-87 MG (Human glioblastoma cells)/MCF7 (Human breast cancer cells)
Fixation: 4% Paraformaldehyde, 15 minutes at room temperature.
Permeabilization: 1x Permeabilization Buffer (eBioscience), 15 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 15 minutes at room temperature.
Antibody dilution buffer: 1x PBS.
Primary antibody: HA724405 (1/1,000) (Red) compared with Rabbit IgG Isotype Control (HA722127, Green), 15 minutes at room temperature.
Secondary antibody: Goat Anti-Rabbit IgG (iFluor™ 488, HA1121), 15 minutes at room temperature.
Low expression: MCF7 cell (PMID: 31197235).
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Immunoprecipitation (IP)
ALDH1A3 was immunoprecipitated in 0.2 mg U-87 MG (Human glioblastoma cells) cell lysate with HA724405 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA724405 at 1/5,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.
Lane 1: U-87 MG cell lysate (input)
Lane 2: HA724405 IP in U-87 MG cell lysate
Lane 3: Rabbit IgG instead of HA724405 in U-87 MG cell lysate
Exposure time: 14 seconds
Blocking: 5% NFDM/TBST, 1 hour at room temperature
Primary dilution: HA724405, 1/5,000 in primary antibody dilution buffer (K1803), 2 hours at room temperature
Predicted band size: 56 kDa
Observed band size: 50-56 kDa
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