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☑ Relative expression (RE)
Western blot analysis of MyoD1 on different lysates with Rabbit anti-MyoD1 antibody (HA724450) at 1/5,000 dilution.
Lane 1: RD (Human malignant embryonal rhabdomyosarcoma cells) cell lysate (15 µg/Lane)
Lane 2: HEK-293 (Human embryonic kidney cells) cell lysate (15 µg/Lane)
Lane 3: HeLa (Human cervical adenocarcinoma cells) cell lysate (15 µg/Lane)
Lane 4: C2C12 (Mouse myoblasts) cell lysate (15 µg/Lane)
Lane 5: Rat embryo tissue lysate (30 µg/Lane)
Exposure time: 1 minute 2 seconds; ECL: K1801
Negative expression of MyoD1 protein in HEK-293 cells is consistent with the predicted expression pattern.
HeLa is negative control (PMID: 17028574, PMID: 2123467).
Blocking: 5% NFDM/TBST, 1 hour at room temperature
Primary antibody: HA724450, 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: 35 kDa
Observed band size: 45 kDa
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☑ Relative expression (RE)
Application: Immunocytochemistry (IF-cell)
Species: Human
Sample: RD (Human malignant embryonal rhabdomyosarcoma cells)/HeLa (Human cervix adenocarcinoma epithelial cell)
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: HA724450, 1/500, overnight at 4°C.
Secondary antibody: Goat Anti-Rabbit IgG (iFluor™ 488, HA1121), 45 minutes at room temperature.
Counterstain: Beta tubulin (M120505-2, red), 1/100, overnight at 4℃. The Nuclear counterstain was DAPI (Blue).
Negative control: HeLa (PMID: 17028574, PMID: 2123467).
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Application: Immunohistochemistry (IHC-P)
Species: Rat
Tissue: Embryo
Sample: Paraffin-embedded section
Antigen retrieval: Heat-mediated, citrate buffer (pH 6.0), pressure cooker, 2 minutes.
Wash buffer: 1× TBST
Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.
Primary antibody: HA724450, 1/200, 1 hour at room temperature.
Secondary antibody: HA1119, 20 minutes at room temperature.
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Application: Immunohistochemistry (IHC-P)
Species: Mouse
Tissue: Embryo
Sample: Paraffin-embedded section
Antigen retrieval: Heat-mediated, citrate buffer (pH 6.0), pressure cooker, 2 minutes.
Wash buffer: 1× TBST
Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.
Primary antibody: HA724450, 1/200, 1 hour at room temperature.
Secondary antibody: HA1119, 20 minutes at room temperature.
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☑ Relative expression (RE)
Application: Flow Cytometry (Intra)
Species: Human
Sample: RD (Human malignant embryonal rhabdomyosarcoma cells)/HeLa (Human cervix adenocarcinoma epithelial cell)
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: HA724450 (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.
Negative control: HeLa (PMID: 17028574, PMID: 2123467).
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Immunoprecipitation (IP)
MyoD1 was immunoprecipitated in 0.2 mg RD (Human malignant embryonal rhabdomyosarcoma cell) cell lysate with HA724450 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA724450 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: RD cell lysate (input)
Lane 2: HA724450 IP in RD cell lysate
Lane 3: Rabbit IgG instead of HA724450 in RD cell lysate
Exposure time: 1 minute
Blocking: 5% NFDM/TBST, 1 hour at room temperature
Primary dilution: HA724450, 1/5,000 in primary antibody dilution buffer (K1803), 2 hours at room temperature
Predicted band size: 35 kDa
Observed band size: 45 kDa
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