- PA5-16327 - Invitrogen Antibodies HSP90AB1 antibody

Estrada-Bernal A, Sanford SD, Sosa LJ, Simon GC, Hansen KC, Pfenninger KH
PloS one 2012;7(2):e31858

Sarkar AA, Zohn IE
The Journal of cell biology 2012 Mar 19;196(6):789-800

Xu Y, Qian H, Feng X, Xiong Y, Lei M, Ren Z, Zuo B, Xu D, Ma Y, Yuan H
Journal of proteomics 2012 Apr 3;75(7):2093-108

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of HSP90 was performed by loading 30 µg of THP-1 whole cell lysate per well onto an SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% non-fat dry milk in TBST for 1 hour at room temperature. The membrane was probed with an Hsp90 polyclonal antibody (Product # PA5-16327) at a dilution of 1:1000 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated goat anti-rabbit IgG secondary antibody at a dilution of 1:40,000 for 1 hour at room temperature. Detection was performed using ECL substrate. Data courtesy of the Innovators Program.

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), NIH/3T3 (Lane 2), PC-3 (Lane 3), LNCaP (Lane 4), K-562 (Lane 5), A549 (Lane 6), HT-1080 (Lane 7) and HEK 293 (Lane 8). The blot was probed with Rabbit Anti-HSP90 Polyclonal Antibody (Product # PA5-16327, 1 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 90 kDa band corresponding to HSP90 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: Immunofluorescent analysis of Heat Shock Protein 90 beta (green) in HeLa cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA (Product # 37525) for 15 minutes at room temperature. Cells were probed without (left panel) or with (right panel) a Heat Shock Protein 90 beta polyclonal antibody (Product # PA5-16327) at a dilution of 1:200 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-rabbit IgG secondary antibody (Product # 35552) at a dilution of 1:400 for 30 minutes at room temperature. F-Actin (red) was stained with DyLight 554 Phalloidin (Product # 21834) and nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan or a ToxInsight Instrument at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Hsp90 beta (green) HEK293T cells. Cells fixed with 4% formaldehyde were permeabilized and blocked with 1X PBS containing 5% BSA and 0.3% Triton X-100 for 1 hour at room temperature. Cells were probed with a Hsp90 beta polyclonal antibody (Product # PA5-16327) at a dilution of 1:100 overnight at 4°C in 1X PBS containing 1% BSA and 0.3% Triton X-100, washed with 1X PBS, and incubated with a fluorophore-conjugated goat anti-rabbit IgG secondary antibody at a dilution of 1:200 for 1 hour at room temperature. Nuclei (blue) were stained with DAPI. Images were taken on a Leica DM1000 microscope at 40X magnification. Data courtesy of the Innovators Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of Heat Shock Protein 90 beta using Heat Shock Protein 90 beta Polyclonal Antibody (Product # PA5-16327) on Native Mouse MAD109 Cells.

PA5-16327