- PA5-21662 - Invitrogen Antibodies MSLN antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Mesothelin Polyclonal Antibody (Product # PA5-21662) and a ~50 kDa band corresponding to Mesothelin was observed in HeLa and NIH: OVCAR-3 and was absent in SH-SY5Y, PC-3, A549 and PANC-1; along with an uncharacterised band (*) at ~60 kDa across cell lines tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), NIH:OVCAR-3 (Lane 2), SH-SY5Y (Lane 3), PC-3 (Lane 4), A549 (Lane 5) and PANC-1 (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). 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 (1:2000 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 SuperSignal™ West Dura Extended Duration Substrate (Product # 34076).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using Mesothelin Polyclonal Antibody (Product # PA5-21662). Sample (30 µg of whole cell lysate). A: 293T. 7.5% SDS PAGE. Mesothelin Polyclonal Antibody (Product # PA5-21662) diluted at 1:1,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Mesothelin was achieved by transfecting NIH:OVCAR-3 with Mesothelin specific siRNAs (Silencer® select Product # S19967, S19968). Western blot analysis (Fig. a) was performed using Whole cell extracts from the Mesothelin knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Mesothelin Polyclonal Antibody (Product # PA5-21662, 1:2000 dilution ) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Mesothelin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Mesothelin was performed using 70% confluent log phase NIH:OVCAR-3 cells. The cells were fixed with 4% paraformaldehyde for 5 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with Mesothelin Polyclonal Antibody (Product # PA5-21662) at 1:200 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), 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 Nucleus and cytoplasmic localization. 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)
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Experimental details: Immunohistochemical analysis of paraffin-embedded human serous ovarian cancer, using MSLN (Product # PA5-21662) antibody at 1:100 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

PA5-21662