- MA5-25949 - Invitrogen Antibodies AHSG antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Fetuin A was achieved by transfecting Hep G2 cells treated with Protein Transport Inhibitor with Fetuin A specific siRNAs (Silencer® select Product # S1201, S1203). Western blot analysis (Fig. a) was performed using whole cell extracts from the Fetuin A knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2), and untransfected cells (lane 1). The blot was probed with Fetuin A Monoclonal Antibody (OTI2H2) (Product # MA5-25949, 1:1000) and Goat anti Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000). Densitometric analysis of this western blot is shown in the histogram (Fig. b). A decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Fetuin A.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Fetuin A Monoclonal Antibody (OTI2H2) (Product # MA5-25949) and 50 kDa band corresponding to Fetuin A was observed in Hep G2 treated cells but not in A549 cells as reported in literature. whole cell extracts (30 µg lysate) of Hep G2 (Lane 1), Hep G2 treated with Protein Transport Inhibitor (1X) for 4hrs (Lane 2), A549 (Lane 3), and A549 treated with Protein Transport Inhibitor (1X) for 4hrs (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein 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-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)
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Experimental details: Immunofluorescence analysis of Fetuin A was performed using Hep G2 and A549 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 Anti-Fetuin A Monoclonal Antibody (OTI2H2) (Product # MA5-25949) at 1:100 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 conjugate (Product # A32766) 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 localization of Fetuin A in Hep G2 cells but not in A549 cells (Panel e) which is reported to be negative for Fetuin A expression. 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)
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Experimental details: Immunohistochemistry was performed on paraffin-embedded adenocarcinoma of human ovary tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a AHSG monoclonal antibody (Product # MA5-25949).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on paraffin-embedded carcinoma of human thyroid tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a AHSG monoclonal antibody (Product # MA5-25949).

MA5-25949