Anti-Hsp27 antibody [RM1148]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp27 antibody [RM1148] (AB317707)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Hsp27 with ab317707 at 1/2000 (0.243 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : no staining on human liver. The section was incubated with ab317707 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp27 antibody [RM1148] (AB317707)

Immunohistochemical analysis of paraffin-embedded Human stomach tissue labeling Hsp27 with ab317707 at 1/2000 (0.243 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on smooth muscle in human stomach. The section was incubated with ab317707 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Hsp27 antibody [RM1148] (AB317707)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HSPB1 KO HAP1 (HSPB1 knockout human chronic myelogenous leukemia near-haploid cell) cells labelling Hsp27 with ab317707 at 1/500 (0.97 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image displaying cytoplasmic staining in wildtype HAP1 cells, and no staining in HSPB1 knockout HAP1 cells (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Hsp27 antibody [RM1148] (AB317707)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized parental HAP1(human chronic myelogenous leukemia near-haploid cell, Left) / HSPB1 knockout HAP1(Right) cells labelling Hsp27 with ab317707 at 1/5000 dilution (0.01 ug)/Red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IP

Supplier Data

Immunoprecipitation - Anti-Hsp27 antibody [RM1148] (AB317707)

Hsp27 was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab317707 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317707 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : ab317707 IP in HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab317707 in HeLa whole cell lysate

All lanes:

Immunoprecipitation - Anti-Hsp27 antibody [RM1148] (ab317707) at 1/30 dilution

All lanes:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 3s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp27 antibody [RM1148] (AB317707)

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling Hsp27 with ab317707 at 1/4000 (0.121 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on smooth muscle in mouse colon. The section was incubated with ab317707 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp27 antibody [RM1148] (AB317707)

Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling Hsp27 with ab317707 at 1/4000 (0.121 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : weak staining on vascular smooth muscle in rat liver. The section was incubated with ab317707 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp27 antibody [RM1148] (AB317707)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Hsp27 with ab317707 at 1/4000 (0.121 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : no staining on mouse liver. The section was incubated with ab317707 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Hsp27 antibody [RM1148] (AB317707)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized C2C12 (mouse myoblast) cells labelling Hsp27 with ab317707 at 1/500 (0.97 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing cytoplasmic staining in C2C12 cells (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp27 antibody [RM1148] (AB317707)

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling Hsp27 with ab317707 at 1/4000 (0.121 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on smooth muscle in rat colon. The section was incubated with ab317707 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Hsp27 antibody [RM1148] (AB317707)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PC-12 (rat adrenal gland pheochromocytoma cell) cells labelling Hsp27 with ab317707 at 1/500 (0.97 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing cytoplasmic staining in PC-12 cells (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-Hsp27 antibody [RM1148] (AB317707)

Hsp27 was immunoprecipitated from 0.35 mg PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate with ab317707 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317707 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate
Lane 2 : ab317707 IP in PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab317707 in PC-12 whole cell lysate

All lanes:

Immunoprecipitation - Anti-Hsp27 antibody [RM1148] (ab317707) at 1/30 dilution

All lanes:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 10s

• IP

Supplier Data

Immunoprecipitation - Anti-Hsp27 antibody [RM1148] (AB317707)

Hsp27 was immunoprecipitated from 0.35 mg C2C12 (mouse myoblast) whole cell lysate with ab317707 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317707 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : C2C12 (mouse myoblast) whole cell lysate
Lane 2 : ab317707 IP in C2C12 (mouse myoblast) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab317707 in C2C12 whole cell lysate

All lanes:

Immunoprecipitation - Anti-Hsp27 antibody [RM1148] (ab317707) at 1/30 dilution

All lanes:

C2C12 (mouse myoblast) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 3s

• WB

Supplier Data

Western blot - Anti-Hsp27 antibody [RM1148] (AB317707)

Low expression : brain, cerebellum.

The expression molecular weight observed is consistent with what has been described in the literature (PMID : 36690850 and PMID : 26675522).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Hsp27 antibody [RM1148] (ab317707) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Rat brain tissue lysate at 20 µg

Lane 3:

Mouse colon tissue lysate at 20 µg

Lane 4:

Mouse heart tissue lysate at 20 µg

Lane 5:

Rat heart tissue lysate at 20 µg

Lane 6:

Rat colon tissue lysate at 20 µg

Lane 7:

Human cerebellum tissue lysate at 20 µg

Lane 8:

Human colon tissue lysate at 20 µg

Lane 9:

C2C12 (mouse myoblast) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 23 kDa,27 kDa,36 kDa

false

Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-Hsp27 antibody [RM1148] (AB317707)

The samples were run on a Bis-Tris gel under reducing conditions.

Western blot : Anti-Hsp27 antibody (ab317707) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5](ab8245) loading control staining at 1/20000 dilution, shown in red.

In Western blot, ab317707 was shown to bind specifically to Hsp27. Target of interest was observed at 23-27 kDa in wild-type HAP1 cell lysates (lane 1) with no signal observed at this size in Hsp27 knockout cell line (lane 2). To generate this image, samples were first run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in a fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed in TBS-T, incubated with secondary antibodies Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution for 1 h at room temperature, washed again then imaged.

The expression molecular weight observed is consistent with what has been described in the literature (PMID : 36690850 and PMID : 26675522).

All lanes:

Western blot - Anti-Hsp27 antibody [RM1148] (ab317707) at 1/1000 dilution

Lane 1:

Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 20 µg

Lane 2:

HSPB1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (800CW) and Goat Anti-Mouse IgG H&L (680RD) at 1/20000 dilution

Observed band size: 23 kDa,27 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-Hsp27 antibody [RM1148] (AB317707)

All lanes:

Western blot - Anti-Hsp27 antibody [RM1148] (ab317707) at 1/1000 dilution

All lanes:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 23 kDa

false

Exposure time: 81s