Hsp90 alpha Antibody (monoclonal, 6B5)

• Catalog Number: orb763097
• Category: Antibodies
• Description: Hsp90 alpha Antibody (monoclonal, 6B5)
• Species/Host: Mouse
• Clonality: Monoclonal
• Clone Number: 6B5
• Tested applications: FC, ICC, IF, IHC, WB
• Reactivity: Human, Monkey, Mouse, Rat
• Isotype: Mouse IgG2b
• Immunogen: A synthetic peptide corresponding to a sequence at the C-terminus of human Hsp90 alpha (454-488aa QNRKKLSELLRYYTSASGDEMVSLKDYCTRMKENQ), identical to the related mouse and rat sequences.
• Concentration: Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Dilution range: Western blot, 0.25-0.5μg/ml, Human, Mouse, Rat, Monkey Immunohistochemistry (Paraffin-embedded Section), 2-5μg/ml, Human Immunocytochemistry/Immunofluorescence, 5μg/ml, Human Flow Cytometry, 1-3μg/1x106 cells, Human
• Form/Appearance: Lyophilized
• Conjugation: Unconjugated
• MW: 90 kDa
• UniProt ID: P07900
• Storage: Store at -20˚C for one year from date of receipt. After reconstitution, at 4˚C for one month. It can also be aliquotted and stored frozen at -20˚C for six months. Avoid repeated freeze-thaw cycles.
• Note: For research use only
• Application notes: Tested Species: In-house tested species with positive results. Other applications have not been tested. Optimal dilutions should be determined by end users. Add 0.2ml of distilled water will yield a concentration of 500ug/ml.
• Expiration Date: 12 months from date of receipt.

Western blot analysis of Hsp90 alpha using anti-Hsp90 alpha antibody (orb763097). Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel)/90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30ug of sample under reducing conditions. Lane 1: human HeLa whole cell lysates, Lane 2: human HEK293 whole cell lysates, Lane 3: monkey COS-7 whole cell lysates, Lane 4: human HepG2 whole cell lysates, Lane 5: human A549 whole cell lysates, Lane 6: rat PC-12 whole cell lysates, Lane 7: rat RH35 whole cell lysates, Lane 8: mouse HEPA1-6 whole cell lysates. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/TBS for 1.5 hour at RT. The membrane was incubated with mouse anti-Hsp90 alpha antigen affinity purified monoclonal antibody (Catalog # orb763097) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # orb90502) with Tanon 5200 system. A specific band was detected for Hsp90 alpha at approximately 90KD. The expected band size for Hsp90 alpha is at 90KD.

IHC analysis of Hsp90 alpha using anti-Hsp90 alpha antibody (orb763097). Hsp90 alpha was detected in paraffin-embedded section of human cervical cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml mouse anti-Hsp90 alpha Antibody (orb763097) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # orb90443) with DAB as the chromogen.

IHC analysis of Hsp90 alpha using anti-Hsp90 alpha antibody (orb763097). Hsp90 alpha was detected in paraffin-embedded section of human colon cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml mouse anti-Hsp90 alpha Antibody (orb763097) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # orb90443) with DAB as the chromogen.

IHC analysis of Hsp90 alpha using anti-Hsp90 alpha antibody (orb763097). Hsp90 alpha was detected in paraffin-embedded section of human lung cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml mouse anti-Hsp90 alpha Antibody (orb763097) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # orb90443) with DAB as the chromogen.

IHC analysis of Hsp90 alpha using anti-Hsp90 alpha antibody (orb763097). Hsp90 alpha was detected in paraffin-embedded section of human testis cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml mouse anti-Hsp90 alpha Antibody (orb763097) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # orb90443) with DAB as the chromogen.

IF analysis of Hsp90 alpha using anti-Hsp90 alpha antibody (orb763097). Hsp90 alpha was detected in immunocytochemical section of SiHa cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (orb90553) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5μg/mL mouse anti-Hsp90 alpha Antibody (orb763097) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Flow Cytometry analysis of A549 cells using anti-Hsp90 alpha antibody (orb763097). Overlay histogram showing A549 cells stained with orb763097 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-Hsp90 alpha Antibody (orb763097, 1μg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (5-10μg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1μg/1x10^6) used under the same conditions. Unlabelled sample (Red line) was also used as a control.