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Western blotAntibody data
- Antibody Data
- Antigen structure
- References [2]
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- Validations
- Western blot [1]
- Immunocytochemistry [1]
- Immunohistochemistry [1]
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- Product number
- 14-9499-82 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- alpha-Fetoprotein Monoclonal Antibody (1E8), eBioscience™
- Antibody type
- Monoclonal
- Antigen
- Other
- Description
- Description: This 1E8 monoclonal antibody reacts with human alpha-fetoprotein (AFP). This 70-kDa secretory protein is a member of the albumin gene family. Synthesized by the yolk sac and fetal liver during embryogenesis, AFP protein levels are highest in fetal serum. After birth, serum AFP levels decrease dramatically. In fact, AFP is nearly undetectable in normal adult serum. However, hepatocellular carcinoma and germ cell teratoblastoma, as well as liver regeneration, viral hepatitis, and cirrhosis, leads to elevated AFP serum levels in adults. As such, detection of this protein is frequently used as a diagnostic marker for these conditions. Applications Reported: This 1E8 antibody has been reported for use in western blotting, immunohistochemical staining of formalin-fixed paraffin embedded tissue sections, and immunocytochemistry. Applications Tested: This 1E8 antibody has been tested by western blot on reduced HepG2 cell lysate and by immunohistochemistry on FFPE human liver section using high pH antigen retrieval. The antibody can be used at less than or equal to 5 µg/mL. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. Purity: Greater than 90%, as determined by SDS-PAGE. Aggregation: Less than 10%, as determined by HPLC. Filtration: 0.2 µm post-manufacturing filtered.
- Reactivity
- Human
- Host
- Mouse
- Isotype
- IgG
- Antibody clone number
- 1E8
- Vial size
- 100 µg
- Concentration
- 0.5 mg/mL
- Storage
- 4° C
Submitted references Molecular mechanisms of alpha-fetoprotein gene expression.
Generation of monoclonal antibodies to alpha-fetoprotein and application in solid-phase enzyme immunoassay.
Lazarevich NL
Biochemistry. Biokhimiia 2000 Jan;65(1):117-33
Biochemistry. Biokhimiia 2000 Jan;65(1):117-33
Generation of monoclonal antibodies to alpha-fetoprotein and application in solid-phase enzyme immunoassay.
Kuo CY, Fu J, Yeh MY, Su SL, Lee CY
Biotechnology and applied biochemistry 1989 Feb;11(1):96-104
Biotechnology and applied biochemistry 1989 Feb;11(1):96-104
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-AFP Monoclonal Antibody (Product # 14-9499-82) 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 (5 µg/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 AFP was performed using 70% confluent log phase Hep G2 and MDA-MB-231 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with AFP Mouse Monoclonal Antibody (Product # 14-9499-82) at 5 µg/mL in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor 488 (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 composite image showing cytoplasmic and golgi staining of AFP in Hep G2 cells. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification..
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunohistochemistry of formalin-fixed paraffin embedded human liver with 5 µg/mL Mouse IgG1 K Isotype Control (left) or 5 µg/mL Anti-Human alpha-Fetoprotein Purified followed by Anti-Mouse IgG Biotin and DAB visualization.Nuclei are counterstained with hematoxylin.
