Anti-HSPA2 Antibody (monoclonal, 4A4)

• Catalog Number: orb443135
• Category: Antibodies
• Description: Anti-HSPA2 Antibody (monoclonal, 4A4). Tested in Flow Cytometry, IF, IHC, ICC, WB applications. This antibody reacts with Human, Mouse, Rat.
• Species/Host: Mouse
• Clonality: Monoclonal
• Clone Number: 4A4
• Tested applications: FC, ICC, IF, IHC, WB
• Reactivity: Human, Mouse, Rat
• Isotype: Mouse IgG1
• Immunogen: A synthetic peptide corresponding to a sequence at the C-terminus of human HSPA2, identical to the related mouse and rat sequences.
• Antibody Type: Primary Antibody
• Concentration: Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Form/Appearance: Lyophilized
• Conjugation: Unconjugated
• MW: 70 kDa
• UniProt ID: P54652
• Storage: Maintain refrigerated at 2-8°C for up to 2 weeks. For long term storage store at -20°C in small aliquots to prevent freeze-thaw cycles.
• Alternative names: Heat shock-related 70 kDa protein 2; Heat shock 70
• Note: For research use only
• Application notes: Western blot, 0.1-0.5μg/ml Immunohistochemistry (Paraffin-embedded Section), 0.5-1μg/ml Immunocytochemistry/Immunofluorescence, 2μg/ml Flow Cytometry (Fixed), 1-3μg/1x106 cells. Add 0.2ml of distilled water will yield a concentration of 500ug/ml
• Expiration Date: 12 months from date of receipt.

Flow Cytometry analysis of PC-3 cells using anti-HSPA2 antibody. Overlay histogram showing PC-3 cells (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-HSPA2 Antibody (1 µg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (5-10 µg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 µg/1x10^6) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

IF analysis of HSPA2 using anti-HSPA2 antibody. HSPA2 was detected in immunocytochemical section of PC-3 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2 µg/mL rabbit anti-HSPA2 Antibody overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit 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.

IHC analysis of HSPA2 using anti-HSPA2 antibody. HSPA2 was detected in paraffin-embedded section of human lung cancer tissue. 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 2 µg/ml mouse anti-HSPA2 Antibody 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) with DAB as the chromogen.

Western blot analysis of HSPA2 using anti-HSPA2 antibody. 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 50 ug of sample under reducing conditions. Lane 1: human Hela whole cell lysate, Lane 2: human MDA-MB-231 whole cell lysate, Lane 3: human COLO-320 whole cell lysate, Lane 4: human PANC-1 whole cell lysate. Lane 5: human HT1080 whole cell lysate, Lane 6: human MDA-MB-453 whole cell lysate, Lane 7: human HepG2 whole cell lysate. 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-HSPA2 antigen affinity purified monoclonal antibody 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 with Tanon 5200 system.

Western blot analysis of HSPA2 using anti-HSPA2 antibody. 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 50 ug of sample under reducing conditions. Lane 1: rat lung tissue lysates, Lane 2: rat liver tissue lysates, Lane 3: rat kidney tissue lysates, Lane 4: rat testicular tissue lysates, Lane 5: mouse lung tissue lysates, Lane 6: mouse liver tissue lysates, Lane 7: mouse kidney tissue lysates, Lane 8: mouse testicular tissue lysates, Lane 9: mouse RAW246.7 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-HSPA2 antigen affinity purified monoclonal antibody 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 with Tanon 5200 system. A specific band was detected for HSPA2 at approximately 70KD. The expected band size for HSPA2 is at 70KD.