- PA5-16341 - Invitrogen Antibodies HSP90AA1 antibody

Song HR, Kim HK, Kim SG, Lim HJ, Kim HY, Han MK
Molecular medicine reports 2021 Jun;23(6)

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot of Heat Shock Protein 90 alpha using Heat Shock Protein 90 alpha Polyclonal Antibody (Product # PA5-16341) on MAD109 Cells.

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 # PA5-16341, 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 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)
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Experimental details: CRISPR-Cas9 mediated genome editing ofHSP90 alpha (as confirmed by next generation sequencing) was achieved by using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR610567_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Fig (a) Western blot analysis of HSP90 alpha was performed by loading 30 µg of HeLa Wild Type (Lane 1), HeLa Cas9 (Lane 2) and HeLa Cas9 cells transduced with HSP90 alpha Lentiviral sgRNA (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein 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 Anti-HSP90 alpha Polyclonal Antibody (Product # PA5-16341) using 1 µg/mL dilution and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036 1:6,000 dilution). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). A reduced signal in sgRNA transduced cells using the LentiArray™ CRISPR product line confirms that antibody is specific toHSP90 alpha (Fig (b)).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of HSP90 alpha was achieved by transfecting HeLa cells with HSP90 alpha specific siRNAs (Silencer® select Product # s6994 and s6995). Western blot analysis (Fig a) was performed using Membrane extract from the HSP90 alpha knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-HSP90 alpha Rabbit Polyclonal Antibody (Product # PA5-16341, 1 µg/mL) and 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). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to HSP90 alpha.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Heat Shock Protein 90 alpha (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 alpha polyclonal antibody (Product # PA5-16341) 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)
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Experimental details: Formalin-fixed, paraffin-embedded human breast carcinoma stained with Heat Shock Protein 90lpha antibody using peroxidase-conjugate and AEC chromogen. Note cytoplasmic and nuclear staining of tumor cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of Heat Shock Protein 90 alpha using Heat Shock Protein 90 alpha Polyclonal Antibody (Product # PA5-16341) on Native Mouse MAD109 Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3. Threonine (Thr5/7) phosphorylation of HSP90alpha decreases in LIF-stimulated mESCs. (A) Following 24-h culture in the absence of LIF, mESCs were treated with vehicle or LIF for 20 min and analyzed by western blotting with antibodies for phosphorylated-HSP90alpha (Thr5/7), HSP90alpha and beta-actin. (B) Densitometric analysis. Data are presented as the mean +- SEM of three experiments. *P

PA5-16341