- PA1-963 - Invitrogen Antibodies PSMA1 antibody

Liu BQ, Meng X, Li C, Gao YY, Li N, Niu XF, Guan Y, Wang HQ
Experimental & molecular medicine 2011 Sep 30;43(9):487-93

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot of 20S C2 subunit in CHO cell extract using Product # PA1-963.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of MDA-MB-231 (Lane 1), MCF7 (Lane 2), PC-3 (Lane 3), Hep G2 (Lane 4) and Jurkat (Lane 5).The blots were probed with PSMA1 Rabbit Polyclonal Antibody (Product # PA1-963, 2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 30 kDa band corresponding to PSMA1 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX), 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)
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Experimental details: The knockdown of PSMA1 was achieved by transfecting Hep G2 with PSMA1 specific siRNAs (Silencer® select Product # s11328, s11329). Western blot analysis (Fig. a) was performed using whole-cell extracts from the PSMA1 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2), and untransfected cells (lane 1). The blot was probed with PSMA1 Polyclonal Antibody (Product # PA1-963, 2 µg/mL concentration) 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 the histogram (Fig. b). The decrease in signal upon siRNA mediated knockdown confirms that the antibody is specific to PSMA1. An uncharacterized band (*) was also observed at ~45 kDa that showed no change upon siRNA transfection.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of PSMA1 was performed using 70% confluent log phase MDA-MB-231 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 PSMA1 Rabbit Polyclonal Antibody (Product # PA1-963) 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 Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e shows the control without primary antibody. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: RNA immunoprecipitation (RIP) western of PSMA1 was performed on K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 µg of PSMA1 polyclonal antibody (Product # PA1-963) rotating 60 min at RT. The immune complexes were captured on 625 µg of anti-rabbit coated Dynabeads (Product # 11204D), washed extensively, and eluted with NuPAGE™ LDS Sample Buffer (Product # NP0007). Samples were resolved onto NuPAGE™ 4-12% Bis-Tris gel (Product # NP0335BOX). Lanes 1 and 3 are input and lanes 2 and 4 are IP. Proteins were transferred to PVDF membrane (Product # IB23001). Membrane was blocked in 5% milk. Target was detected using a PSMA1 polyclonal antibody (Product # PA1-963) at a dilution of 1:2000, followed by a 1:4000 dilution of secondary antibody. Chemiluminescent detection was performed using ECL Western Blotting Substrate (Product # 32106). Data courtesy of the Yeo lab as part of the ENCODE project (www.encodeproject.org).

PA1-963