Hsc70 Antibody(monoclonal, 3B6)

• Catalog Number: orb623830
• Category: Antibodies
• Description: Hsc70 Antibody(monoclonal, 3B6)
• Species/Host: Mouse
• Clonality: Monoclonal
• Clone Number: 3B6
• Tested applications: FC, ICC, IF, IHC, WB
• Reactivity: Human, Mouse, Rat
• Isotype: Mouse IgG2b
• Immunogen: E.coli-derived human Hsc70 recombinant protein (Position: Q520-A614). Human Hsc70 shares 98.9% amino acid (aa) sequence identity with both mouse and rat Hsc70.
• Concentration: Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Form/Appearance: Lyophilized
• Conjugation: Unconjugated
• MW: 71 kDa
• UniProt ID: P11142
• Sensitivity: 1.54 ng/ml
• 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 cognate 71 kDa protein; Heat shock 70 k
• 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 500μg/ml.
• Expiration Date: 12 months from date of receipt.

Flow Cytometry analysis of HEPA1-6 cells using anti-Hsc70 antibody (orb623830). Overlay histogram showing HEPA1-6 cells stained with orb623830 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-Hsc70 Antibody (orb623830, 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 Hsc70 using anti-Hsc70 antibody (orb623830). 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 50ug of sample under reducing conditions. Lane 1: human HEK293 whole cell lysates; Lane 2: human HeLa whole cell lysates; Lane 3: human Jurkat whole cell lysates; Lane 4: human A549 whole cell lysates; Lane 5: human HL-60 whole cell lysates; Lane 6: rat PC-12 whole cell lysates; Lane 7: human RAW264.7 whole cell lysates; Lane 8: mouse NIH/3T3 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-Hsc70 antigen affinity purified monoclonal antibody (Catalog # orb623830) 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 Hsc70 at approximately 71KD. The expected band size for Hsc70 is at 71KD.

IHC analysis of Hsc70 using anti-Hsc70 antibody (orb623830). Hsc70 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 1μg/ml mouse anti-Hsc70 Antibody (orb623830) 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 Hsc70 using anti-Hsc70 antibody (orb623830). Hsc70 was detected in paraffin-embedded section of human rectal cancer (lymph node) 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 1μg/ml mouse anti-Hsc70 Antibody (orb623830) 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 Hsc70 using anti-Hsc70 antibody (orb623830). Hsc70 was detected in paraffin-embedded section of human rectal cancer (lymph node) 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 1μg/ml mouse anti-Hsc70 Antibody (orb623830) 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 Hsc70 using anti-Hsc70 antibody (orb623830). Hsc70 was detected in paraffin-embedded section of human tonsil 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 1μg/ml mouse anti-Hsc70 Antibody (orb623830) 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 Hsc70 using anti-Hsc70 antibody (orb623830). Hsc70 was detected in paraffin-embedded section of rat spleen 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 1μg/ml mouse anti-Hsc70 Antibody (orb623830) 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 Hsc70 using anti-Hsc70 antibody (orb623830). Hsc70 was detected in immunocytochemical section of A549 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 2μg/mL mouse anti-Hsc70 Antibody (orb623830) overnight at 4°C. DyLight?594 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-Hsc70 antibody (orb623830). Overlay histogram showing A549 cells stained with orb623830 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-Hsc70 Antibody (orb623830, 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 K562 cells using anti-Hsc70 antibody (orb623830). Overlay histogram showing K562 cells stained with orb623830 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-Hsc70 Antibody (orb623830, 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.