MA5-14666

antibody from Invitrogen Antibodies

Targeting: AFP FETA, HPAFP

Provider product page for MA5-14666

Western blot

ELISA

Immunocytochemistry

Immunoprecipitation

Radioimmunoassay

Other assay

Antibody data

Antibody Data
Antigen structure
References [1]
Comments [0]
Validations
Western blot [5]
Immunocytochemistry [3]
Other assay [1]

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Product number: MA5-14666 - Provider product page
Provider: Invitrogen Antibodies
Product name: AFP Monoclonal Antibody (P5B8)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: MA5-14666 can be used for immunofluorescence analysis of AFP in the endoderm derived from human embryonic stem cells. By Western blot, MA5-14666 detects endogenous AFP protein in the early hepatocyte-like cells derived from human embryonic stem cells. Product MA514666 is a smaller package size of MIA1305 (formerly sold as a Seradyn product).
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: P5B8
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: Maintain refrigerated at 2-8°C for up to 6 months. For long term storage store at -20°C

AFP protein structure - MA5-14666 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 # MA5-14666) 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 # MA5-14666) 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 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). GAPDH was detected using a GAPDH rabbit polyclonal antibody, HRP conjugate (Product # PA1-987-HRP), at a dilution of 1:1000 for at least 1 hour at room temperature.

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 # MA5-14666) 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: Western blot analysis was performed on whole cell extracts (30 µg lysate) of Hep G2 (Lane 1), Hep G2 Conditioned Media (Lane 2) and HeLa Conditioned media (Lane 3). The blot was probed with Anti- AFP Mouse Monoclonal Antibody (Product # MA5-14666, 1:1000 dilution) and detected by chemiluminescence using Goat anti Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 ug/ml, 1:4000 dilution). A 68 kDa band corresponding to AFP was detected in Hep G2 conditioned media but not in HeLa conditioned media which is negative for AFP expression.

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 # MA5-14666, 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 # MA5-14666, 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of AFP was performed using 70% confluent log phase Hep G2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with AFP Mouse Monoclonal Antibody (Product # MA5-14666) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic and Golgi localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.