MIA1305

antibody from Invitrogen Antibodies

Targeting: AFP FETA, HPAFP

Antibody data

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Validation data

Product number: MIA1305 - Provider product page
Provider: Invitrogen Antibodies
Product name: AFP Monoclonal Antibody (P5B8)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: MIA1305 can be used for immunofluorescence analysis of AFP in the endoderm derived from human embryonic stem cells. By Western blot, MIA1305 detects endogenous AFP protein in the early hepatocyte-like cells derived from human embryonic stem cells.
Antibody clone number: P5B8
Concentration: 1.0 mg/mL

AFP protein structure - MIA1305 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of alpha-fetoprotein was performed by loading 10 µg of whole cell extract from H9 ESCs, H9 ESC-derived hepatoblasts, ESC-derived hepatocyte-like cells, HepG2 cells, HepaRG cells, and primary human hepatocytes in reducing conditions and 10 µL Spectra Multicolor Broad Range Protein Ladder (Product # 26634) per well onto a Novex Bolt® 4-12% Bis-Tris Plus Gel with Bolt® MOPS running buffer and Bolt® Antioxidant. Proteins were transferred to a PVDF membrane using the iBlot 2 Dry Blotting System (Product # IB21001), and blocked with 5% non-fat milk in TBST for 1 hour at room temperature. alpha-Fetoprotein was detected at 67 kDa using an alpha-fetoprotein monoclonal antibody (Product # MIA1305) at a concentration of 1 µg/mL in 5% non-fat milk in TBST overnight at 4°C on a rocking platform, followed by a goat anti-mouse secondary antibody (Product # A24518) at a dilution of 1:10,000 for 1 hour at room temperature. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of alpha-fetoprotein was performed by loading 10 µg of whole cell extract from H9 ESCs, H9 ESC-derived hepatoblasts, ESC-derived hepatocyte-like cells, HepG2 cells, HepaRG cells, and primary human hepatocytes in reducing conditions and 10 µL Spectra Multicolor Broad Range Protein Ladder (Product # 26634) per well onto a Novex Bolt® 4-12% Bis-Tris Plus Gel with Bolt® MOPS running buffer and Bolt® Antioxidant. Proteins were transferred to a PVDF membrane using the iBlot 2 Dry Blotting System (Product # IB21001), and blocked with 5% non-fat milk in TBST for 1 hour at room temperature. alpha-Fetoprotein was detected at 67 kDa using an alpha-fetoprotein monoclonal antibody (Product # MIA1305) at a concentration of 1 µg/mL in 5% non-fat milk in TBST overnight at 4°C on a rocking platform, followed by a goat anti-mouse secondary antibody (Product # A24518) at a dilution of 1:10,000 for 1 hour at room temperature. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of alpha-fetoprotein was performed by loading 40 µg of the indicated whole cell lysates and 5 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a 4-20% Tris-Glycine polyacrylamide gel (Product # WT4201BX10). Proteins were transferred to a nitrocellulose membrane using the G2 Blotter (Product # 62288), and blocked with 5% BSA in TBST for 1 hour at room temperature. Alpha-fetoprotein was detected at ~67 kDa using a alpha-fetoprotein mouse monoclonal antibody (Product # MIA1305) at a concentration of 2 µg/mL in blocking buffer overnight at 4C on a rocking platform, followed by a Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177) at a dilution of 1:1000 for at least 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34078).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of alpha-fetoprotein was performed by loading 20 µg of the indicated whole cell lysates and 5 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a 4-20% Tris-Glycine polyacrylamide gel (Product # WT4202BX10). Proteins were transferred to a nitrocellulose membrane using the G2 Blotter (Product # 62288), and blocked with 5% BSA in TBST for 1 hour at room temperature. Alpha-fetoprotein was detected at ~67 kDa using a alpha-fetoprotein mouse monoclonal antibody (Product # MIA1305) at a concentration of 2 µg/mL in blocking buffer for 1 hour at room temperature on a rocking platform, followed by a Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177) at a dilution of 1:1000 for at least 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34078).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of alpha-fetoproprotein (AFP) (green) in the endoderm derived from human ES cells. Embryoid bodies (EBs) were generated from the H9 embryonic stem cell line (WiCell Research Institute, WA09) using Gibco® KnockOut Serum Replacement. After four days in suspension culture, EBs were plated on Geltrex-coated tissue culture-treated polystyrene plates and continuously cultured for 21 days. EB cultures were then fixed and permeabilized according to the 3-Germ Layer Immunocytochemistry Kit (Product # A25538) and stained with anti-alpha-fetoprotein (Product # MIA1305, 1:200 dilution, 5 uL/mL final) at 4°C overnight. Secondary staining was completed using Alexa Fluor 488-conjugated anti-mouse IgG (Product # A-11001) and DAPI (Product # D1306) for nuclear DNA (blue) for 1 h at room temperature. Stained wells were imaged at 40X using the EVOS® FL Auto Imaging System.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of alpha-fetoprotein (green) in the endoderm derived from human ES cells. Embryoid bodies (EBs) were generated from the H9 embryonic stem cell line (WiCell Research Institute, WA09) using Gibco® KnockOut Serum Replacement. After four days in suspension culture, EBs were plated on Geltrex-coated tissue culture-treated polystyrene plates and continuously cultured for 21 days. EB cultures were then fixed and permeabilized according to the 3-Germ Layer Immunocytochemistry Kit (Product # A25538) and blocked with 3% BSA for 30 minutes at room temperature. Cells were stained with anti-alpha-fetoprotein (Product # MIA1305, 1:200 dilution, 5 µg/mL final concentration) at 4°C overnight, and then incubated with a Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:1000 for at least 30 minutes at a room temperature in the dark (green). Nuclei (blue) were stained with Hoechst 33342 (Product # 62249). Stained cells were imaged at 40X using the EVOS® FL Auto Imaging System.