HSPA4 Antibody / Heat shock protein family A member 4 (FY13366)

Western blot analysis of HSPA4 using anti-HSPA4 antibody. Lane 1: human 293T whole cell lysates, Lane 2: human K562 whole cell lysates, Lane 3: rat brain tissue lysates, Lane 4: rat C6 whole cell lysates, Lane 5: mouse brain tissue lysates, Lane 6: mouse NIH/3T3 whole cell lysates, Lane 7: mouse Raw264.7 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 rabbit anti-HSPA4 antibody at 0.5 ug/ml overnight at 4oC, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal was developed using enhanced chemiluminescent. A dominant band is observed at approximately 105-110 kDa, which is the expected migration pattern for HSPA4. Although the theoretical molecular weight of HSPA4 is ~94 kDa, members of the Hsp110 family, including HSPA4, are well known to migrate 10-20 kDa heavier due to acidic domains, extended disordered regions, and phosphorylation, which reduce SDS binding and cause anomalous gel mobility.

Western blot analysis of HSPA4 using anti-HSPA4 antibody. Lane 1: human 293T whole cell lysates, Lane 2: human K562 whole cell lysates, Lane 3: rat brain tissue lysates, Lane 4: rat C6 whole cell lysates, Lane 6: mouse brain tissue lysates, Lane 7: mouse NIH/3T3 whole cell lysates, Lane 8: mouse Raw264.7 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 rabbit anti-HSPA4 antibody at 0.25 ug/ml overnight at 4oC, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-DyLight 647 Conjugated secondary antibody at a dilution of 1:2000 for 1.5 hour at RT. A dominant band is observed at approximately 105-110 kDa, which is the expected migration pattern for HSPA4. Although the theoretical molecular weight of HSPA4 is ~94 kDa, members of the Hsp110 family, including HSPA4, are well known to migrate 10-20 kDa heavier due to acidic domains, extended disordered regions, and phosphorylation, which reduce SDS binding and cause anomalous gel mobility.

Immunohistochemical staining of HSPA4 using anti-HSPA4 antibody. HSPA4 was detected in a paraffin-embedded section of human colon cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 ug/ml rabbit anti-HSPA4 antibody overnight at 4oC. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37oC. The tissue section was developed using an HRP secondary and DAB substrate.

Immunohistochemical staining of HSPA4 using anti-HSPA4 antibody. HSPA4 was detected in a paraffin-embedded section of mouse ovary tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 ug/ml rabbit anti-HSPA4 antibody overnight at 4oC. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37oC. The tissue section was developed using an HRP secondary and DAB substrate.

Flow Cytometry analysis of 293T cells using anti-HSPA4 antibody. Overlay histogram showing 293T cells stained with (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-HSPA4 antibody (1 ug/million cells) for 30 min at 20oC. DyLight 488 conjugated goat anti-rabbit IgG (5-10 ug/million cells) was used as secondary antibody for 30 minutes at 20oC. Isotype control antibody (Green line) was rabbit IgG (1 ug/million cells) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Flow Cytometry analysis of MOLT-4 cells using anti-HSPA4 antibody. Overlay histogram showing MOLT-4 cells stained with (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-HSPA4 antibody (1 ug/million cells) for 30 min at 20oC. DyLight 488 conjugated goat anti-rabbit IgG (5-10 ug/million cells) was used as secondary antibody for 30 minutes at 20oC. Isotype control antibody (Green line) was rabbit IgG (1 ug/million cells) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.