Anti-HSPA2 Antibody Picoband™ (monoclonal, 4A4)

Figure 1. IHC analysis of HSPA2 using anti-HSPA2 antibody (M03474-1).
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 2μg/ml mouse anti-HSPA2 Antibody (M03474-1) 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 # SA1021) with DAB as the chromogen.

Figure 2. Western blot analysis of HSPA2 using anti-HSPA2 antibody (M03474-1).
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 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 (Catalog # M03474-1) 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 # EK1001) with Tanon 5200 system.

Figure 3. IF analysis of HSPA2 using anti-HSPA2 antibody (M03474-1).
HSPA2 was detected in immunocytochemical section of PC-3 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2μg/mL rabbit anti-HSPA2 Antibody (M03474-1) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) 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.

Figure 4. Flow Cytometry analysis of PC-3 cells using anti-HSPA2 antibody (M03474-1).
Overlay histogram showing PC-3 cells stained with M03474-1 (Blue line).The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-HSPA2 Antibody (M03474-1,1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

Figure 5. Western blot analysis of HSPA2 using anti-HSPA2 antibody (M03474-1).
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: 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 (Catalog # M03474-1) 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 # EK1001) with Tanon 5200 system. A specific band was detected for HSPA2 at approximately 70KD. The expected band size for HSPA2 is at 70KD.