- PA5-27715 - Invitrogen Antibodies PYCARD antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of TMS1 using 30 µg of HeLa lysate. Samples were loaded onto a 12% SDS-PAGE gel and probed with a TMS1 polyclonal antibody (Product # PA5-27715) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of TMS1 using A) 30 µg 293T whole cell lysate and B) 30 µg whole cell lysate of 3xFlag-human TMS1-transfected 293T cells. Samples were loaded onto a 12% SDS-PAGE gel and probed with a TMS1 polyclonal antibody (Product # PA5-27715) at a dilution of 1:20000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of PYCARD was performed by separating 30 µg of non-transfected (–) and transfected (+) 293T whole cell extracts by 12% SDS-PAGE. Proteins were transferred to a membrane and probed with a PYCARD Polyclonal Antibody (Product # PA5-27715) at a dilution of 1:1000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of PYCARD was performed by separating 30 µg of whole cell extract by 12% SDS-PAGE. Proteins were transferred to a membrane and probed with a PYCARD Polyclonal Antibody (Product # PA5-27715) at a dilution of 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-PYCARD Polyclonal Antibody (Product # PA5-27715) and a 21kDa band corresponding to Apoptosis-associated speck-like protein containing a CARD was observed across the cell lines tested. Whole cell extracts (30 µg lysate) of MCF7 (Lane 1), THP-1 (Lane 2), HL-60 (Lane 3), K-562 (Lane 4), HeLa (Lane 5), Jurkat (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000) 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: PYCARD Polyclonal Antibody detects TMS1 protein at cytoplasm and nucleus by immunofluorescent analysis. Sample: HCT 116 cells were fixed in 2% paraformaldehyde/culture medium at 37oC for 30 min. Green: TMS1 protein stained by PYCARD Polyclonal Antibody (Product # PA5-27715) diluted at 1:500. Blue: Hoechst 33342 staining. Scale bar = 10 µm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Apoptosis-associated speck-like protein containing a CARD was performed using 70% confluent log phase MCF7 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 45 minutes at room temperature. The cells were labeled with PYCARD Polyclonal Antibody (Product # PA5-27715) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731), (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 merged image showing Nucleus and cytoplasm localization. Panel e represents HeLa showing no expression. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

PA5-27715