- PA5-29356 - Invitrogen Antibodies TGM2 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of TGM2 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR979226_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of TGM2 was performed by loading 30 µg of HeLa Wild Type (Lane 1), HeLa Cas9 (Lane 2) andHela TGM2 KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 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 Anti-TGM2 Polyclonal Antibody (Product # PA5-29356, 1:2,000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:6,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to TGM2. Uncharacterized band was observed in all the samples at 50 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using TGM2 Polyclonal Antibody (Product # PA5-29356). Sample (30 µg of whole cell lysate). Lane A: A549 . 7.5% SDS PAGE. TGM2 Polyclonal Antibody (Product # PA5-29356) diluted at 1:2,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of TGM2 was achieved by transfecting HeLa with TGM2 specific siRNAs (Silencer® select Product # S14087, S14088). Western blot analysis (Fig. a) was performed using Whole cell extracts from the TGM2 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with TGM2 Polyclonal Antibody (Product # PA5-29356,1:2000 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 TGM2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-TGM2 Polyclonal Antibody(Product # PA5-29356) and a 78kDa band corresponding to TGM2 was observed across cell lines tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), A549 (Lane 2), MDA-MB-231 (Lane 3), MCF7 (Lane 4), LnCaP (Lane 5) and DU 145 (Lane 6) 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 (1:2000 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 Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Transglutaminase 2 in methanol-fixed A549 cells using a Transglutaminase 2 polyclonal antibody (Product # PA5-29356) at a 1:500 dilution.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of TGM2 was performed using 70% confluent log phase MDA-MB-231 and MCF7 cells. The cells were fixed with 4% Paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 10 minutes at room temperature. The cells were labeled with TGM2 Polyclonal Antibody (Product # PA5-29356) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Alexa Fluor Plus 488 donkey anti-rabbit IgG secondary antibody -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 dilution). Panel d represents the merged image showing cytoplasmic localization. Panel e represents MCF7 cells having no expression of TGM2. 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 (Paraffin) analysis of TGM2 was performed in paraffin-embedded mouse duodenum tissue using TGM2 Polyclonal Antibody (Product # PA5-29356) at a dilution of 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of paraffin-embedded Mahlarvu xenograft, using Transglutaminase 2 (Product # PA5-29356) antibody at 1:500 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

PA5-29356