- PA5-47036 - Invitrogen Antibodies BIRC2 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis from lysates of HEK293 human embryonic kidney cell line transfected with human cIAP-1 (lane 1), human cIAP-2 (lane 2), or non-transfected (lane 3). PVDF membrane was probed with 0.5 µg/mL Goat Anti-human cIAP-1/HIAP-2 Antigen Affinity-purified Polyclonal Antibody (Product # PA5-47036) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody. For additional reference, lystates of HepG2 human hepatocellular carcinoma cell line and Jurkat human acute T cell leukemia cell line were included. A specific band for cIAP-1/HIAP-2 was detected at approximately 65 kDa (as indicated). This experiment was conducted under reducing conditions.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout validation by Western blot analysis of cIAP1 in lysates of HeLa human cervical epithelial carcinoma parental cell line and cIAP-1/HIAP-2 knockout HeLa cell line (KO). Samples were incubated in cIAP1 polyclonal antibody (Product # PA5-47036) using a dilution of 0.5 µg/mL followed by a HRP-conjugated Anti-Goat IgG secondary antibody. A specific band was detected for cIAP‚1/HIAP‚2 at approximately 68 kDa (as indicated) in the parental HeLa cell line, but is not detectable in knockout HeLa cell line. GAPDH is shown as a loading control. This experiment was conducted under reducing conditions.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of cIAP1 in HEK293 human embryonic kidney cell line transfected with human cIAP-1 (lane 1), human cIAP-2 (lane 2), or non-transfected (lane 3). Samples were incubated in cIAP1 polyclonal antibody (Product # PA5-47036) using a dilution of 0.5 µg/mL followed by a HRP-conjugated Anti-Goat IgG secondary antibody. For additional reference, lystates of HepG2 human hepatocellular carcinoma cell line and Jurkat human acute T cell leukemia cell line were included. A specific band for cIAP‚1/HIAP‚2 was detected at approximately 65 kDa (as indicated). This experiment was conducted under reducing conditions.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of cIAP1 in 0.5 mg/mL lysates of HepG2 human hepatocellular carcinoma cell line. Samples were incubated in cIAP1 polyclonal antibody (Product # PA5-47036) using a dilution of 5 µg/mL followed by HRP-conjugated Anti-Goat IgG at a dilution of 0.0763888888888889. A specific band was detected for cIAP‚1/HIAP‚2 at approximately 66 kDa (as indicated). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of BIRC2 was achieved by transfecting HeLa with BIRC2 specific siRNAs (Silencer® select Product # s1450). Western blot analysis (Fig. a) was performed using modified whole cell extracts (1% SDS) from the BIRC2 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with cIAP1 Polyclonal Antibody (Product # PA5-47036, 0.5 µg/ml) 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 BIRC2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-cIAP1 Polyclonal Antibody (Product # PA5-47036) and a 64 kDa corresponding to BIRC2 was observed across cell lines tested. Modified whole cell extracts (1% SDS) (30 µg lysate) of HeLa (Lane 1), Hep G2 (Lane 2), HCT116 (Lane 3), T-47D (Lane 4), MCF-7 (Lane 5), MDA-MB-231 (Lane 6), A549 (Lane 7), LNCaP (Lane 8) and Jurkat (Lane 9) 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 (0.5 µg/ml) and detected by chemiluminescence with Rabbit anti-Goat IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27014, 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 cIAP1 was performed using 70% confluent log phase HeLa 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 cIAP1 Goat Polyclonal Antibody (Product # PA5-47036) at 5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Rabbit anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Product # A-11078) 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). Panel d represents the merged image showing 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 cIAP1 in immersion fixed paraffin-embedded sections of human lymphoma. Samples were incubated in cIAP1 polyclonal antibody (Product # PA5-47036) using a dilution of 15 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown) and counterstained with hematoxylin (blue). Lower panel shows a lack of labeling if primary antibodies are omitted and tissue is stained only with secondary antibody followed by incubation with detection reagents.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of cIAP1 in immersion fixed paraffin-embedded sections of human lymph node. Samples were incubated in cIAP1 polyclonal antibody (Product # PA5-47036) using a dilution of 10 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown) and counterstained with hematoxylin (blue). Specific staining was localized to lymphocytes.

PA5-47036