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Western blot
Flow cytometryAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [3]
- Immunocytochemistry [1]
- Immunohistochemistry [1]
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- Product number
- MA5-16321 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- AFP Monoclonal Antibody (SP154)
- Antibody type
- Monoclonal
- Antigen
- Purifed from natural sources
- Description
- Heat-mediated antigen retrieval is recommended prior to staining, using a 10mM citrate buffer, pH 6.0, for 10 minutes followed by cooling at room temperature for 20 min. Following antigen retrieval, incubate samples with primary antibody for 10 min at room temperature. A suggested positive control is hepatocellular carcinoma or fetal liver.
- Reactivity
- Human
- Host
- Rabbit
- Isotype
- IgG
- Antibody clone number
- SP154
- Vial size
- 500 µL
- Concentration
- 0.237 mg/mL
- Storage
- Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of HepG2 Cell Lysate using anti-Alpha-Fetoprotein Monoclonal Antibody (Product # MA5-16321). The recommened dilution for this antibody in western blot applications is 1:100.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of HepG2 Cell Lysate using anti-Alpha-Fetoprotein Monoclonal Antibody (Product # MA5-16321). The recommened dilution for this antibody in western blot applications is 1:100.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-AFP Antibody (Product # MA5-16321) and a 68 kDa band corresponding to AFP was observed in Hep G2 cells but not in MDA-MB-231 cells which are reported to be negative. Whole cell extracts (30 µg lysate) of HepG2 (Lane 1), HepG2 treated with 1X protein transport inhibitor for 4 hours (Lane 2) (Product # 00-4980-03), 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 (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 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 # MA5-16321) at 1:200 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 conjugate (Product # A32790) 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 shows MDA-MB-231 cells with no signal. Panel f 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
- Immunohistochemical analysis of Alpha-Fetoprotein using anti-Alpha-Fetoprotein Monoclonal Antibody (Product # MA5-16321) in Hepatocellular Carcinoma Cancer Tissue. The recommened dilution for this antibody in immunohistochemistry applications is 1:100.
