MIA1301

antibody from Invitrogen Antibodies

Targeting: AFP FETA, HPAFP

ELISA

Immunoprecipitation

Radioimmunoassay

Antibody data

Submit

Validation data

Product number: MIA1301 - Provider product page
Provider: Invitrogen Antibodies
Product name: AFP Monoclonal Antibody (F1-6P2A8-P2B9A9)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: MIA1301 can be used for immunofluorescence analysis of AFP in the endoderm derived from human embryonic stem cells. By Western blot, MIA1301 detects endogenous AFP protein in the early hepatocye-like cells derived from human embryonic stem cells. MIA1301 was formerly sold as a Seradyn product.
Reactivity: Human, Mouse
Host: Mouse
Isotype: IgG
Antibody clone number: F1-6P2A8-P2B9A9
Vial size: 1 mg
Concentration: 5 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 - MIA1301 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, primary human hepatocytes, HepaRG cells, HepG2 cells, and H9 ESC-derived hepatocytes-like cells 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 # MIA1301) 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 was performed using membrane enriched extracts (30 µg) of Hep G2 (Lane 1) and Mouse Liver (Lane 2).The blots were probed with Anti-Alpha Fetoprotein Mouse Monoclonal Antibody (Product # MIA1301, 1 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.4 µg/mL, 1:2500 dilution). A ~69 kDa band corresponding to Alpha Fetoprotein was observed across the cell line and tissue tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800).Resolved proteins were then transferred onto a nitrocellulose membrane by iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody using iBind™ Flex Western Starter Kit (Product # SLF2000S). 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 was performed using Anti-AFP Monoclonal Antibody (Product # MIA1301) and a 68 kDa band corresponding to AFP was observed upon treatment with protein transport inhibitor in Hep G2 cells but not in MDA-MB-231 cells which are reported to be negative. Whole cell extracts (30 µg lysate) of Hep G2 (Lane 1), Hep G2 treated with 1X protein transport inhibitor (Product # 00-4980-03) for 4 hours (Lane 2), MDA-MB-231 (Lane 3) and MDA-MB-231 treated with 1X protein transport inhibitor for 4 hours (Product # 00-4980-03) (Lane 4) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1µg/ml) and detected by chemiluminescence with Goat anti- Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005)..

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 # MIA1301, 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: 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 # MIA1301, 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.