Hsp27/HSPB1 Antibody (monoclonal, 3H3)

• Catalog Number: orb570310
• Category: Antibodies
• Description: Hsp27/HSPB1 Antibody (monoclonal, 3H3)
• Species/Host: Mouse
• Clonality: Monoclonal
• Clone Number: 3H3
• Tested applications: FC, ICC, IF, IHC, WB
• Reactivity: Human
• Isotype: Mouse IgG1
• Immunogen: E.coli-derived human Hsp27/HSPB1 recombinant protein (Position: M1-K205). Human Hsp27 shares 83% amino acid (aa) sequence identity with mouse Hsp27.
• Concentration: 0
• Dilution range: Western blot, 0.1-0.5μg/ml Immunohistochemistry (Paraffin-embedded Section), 0.5-1μg/ml Immunocytochemistry/Immunofluorescence, 2μg/ml Flow Cytometry, 1-3μg/1x106 cells
• Form/Appearance: Lyophilized
• Conjugation: Unconjugated
• MW: 27 kDa
• UniProt ID: P04792
• 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.
• Alternative names: Heat shock protein beta-1; HspB1; 28 kDa heat shoc
• Note: For research use only
• Application notes: Tested Species: In-house tested species with positive results. By Heat: Boiling the paraffin sections in 10mM citrate buffer, pH6.0, for 20mins is required for the staining of formalin/paraffin sections. 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 500μg/ml.
• Expiration Date: 12 months from date of receipt.

IHC analysis of HSP27 using anti-HSP27 antibody (orb570310). HSP27 was detected in paraffin-embedded section of human lung cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml mouse anti-HSP27 Antibody (orb570310) 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 HSP27 using anti-HSP27 antibody (orb570310). HSP27 was detected in paraffin-embedded section of human oesophagus squama cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml mouse anti-HSP27 Antibody (orb570310) 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 HSP27 using anti-HSP27 antibody (orb570310). HSP27 was detected in paraffin-embedded section of human placenta tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml mouse anti-HSP27 Antibody (orb570310) 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 HSP27 using anti- HSP27 antibody (orb570310). HSP27 was detected in immunocytochemical section of U20S cell. 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 2μg/mL mouse anti- HSP27 Antibody (orb570310) 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-HSP27 antibody (orb570310). Overlay histogram showing A549 cells stained with orb570310 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-HSP27 Antibody (orb570310, 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.

Flow Cytometry analysis of U20S cells using anti-HSP27 antibody (orb570310). Overlay histogram showing U20S cells stained with orb570310 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-HSP27 Antibody (orb570310, 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.

Western blot analysis of HSP27 using anti-HSP27 antibody (orb570310). 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 30 ug of sample under reducing conditions. Lane 1: human HeLa whole cell lysates, Lane 2: human CACO-2 whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA 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-HSP27 antigen affinity purified monoclonal antibody (Catalog # orb570310) 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 HSP27 at approximately 27 kDa. The expected band size for HSP27 is at 27 kDa.