- PA5-16358 - Invitrogen Antibodies CASP9 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot of Caspase 9 using Caspase 9 Polyclonal Antibody (Product # PA5-16358) on Jurkat Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of Caspase 9 was achieved by transfecting HeLa cells with Caspase 9 specific siRNAs (Silencer® select Product # s2429, s2430). Western blot analysis (Fig. a) was performed using membrane enriched extracts from the Caspase 9 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-Caspase 9 Polyclonal Antibody (Product # PA5-16358, 1 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 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 Caspase 9.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of HeLa (Lane 1), MCF7 (Lane 2), Jurkat (Lane 3), K-562 (Lane 4), HT-29 (Lane 5) and PC-3 (Lane 6). The blot was probed with Anti-Caspase 9 Rabbit Polyclonal Antibody (Product # PA5-16358, 3 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 45 kDa band corresponding to Caspase 9 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of A-431 (Lane 1), U-87 MG (Lane 2), HeLa (Lane 3). HeLa treated with Staurosporine (1uM for 3 hours) (Lane 4) and Jurkat (Lane 5). The blot was probed with Anti-Caspase 9 Polyclonal Antibody (Product # PA5-16358, 1 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 46 kDa band corresponding to Caspase 9 was observed across the cell lines tested and bands at 39 & 37 kDa was also observed corresponding to cleaved Caspase 9 following Staurosporine treatment.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Caspase 9 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 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Caspase 9 Rabbit Polyclonal Antibody (Product # PA5-16358) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Caspase 9 showing staining in the cytoplasm of paraffin-embedded human heart tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Caspase 9 Rabbit Polyclonal Antibody (Product # PA5-16358) diluted in 3% BSA-PBS at a dilution of 1:20 for 1 hour at 37°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of Caspase 9 using Caspase 9 Polyclonal Antibody (Product # PA5-16358) on denatured Human Jurkat Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 7 Western blot analysis: The cell signaling protein expressions of ( a ) BT-474 and ( b ) MDA-MB-231 cells using PBS, PTX alone, TTZ alone, and PTXNR-TTX treatments. The relative intensity of XIAP, actin, cleaved caspase-9, caspase 3, cleaved caspase 3, and cytochrome C is analyzed after 48 h of treatment in ( c ) BT-474 and ( d ) MDA-MB-231 cells using ImageJ/ Fiji. For each protein expression the experiment was replicated at least n = 2-4 times and the average data is presented as mean +- standard deviation. The relative protein expressions are normalized to the untreated control cells and to the housekeeping GAPDH protein expression (hypothesized expression mean, u = 1). The p values for overexpression or downregulation of cytochrome-C, cleaved caspase-3, and XIAP in BT-474 are 0.039, 0.003, and 0.000049, respectively. The p value for the overexpression of cytochrome-C in MDA-MB-231 is 0.047. ***Represents p < 0.05 (for detailed p value calculations, please see SI Table 6 ).

PA5-16358

Antibody data