Anti-Calponin 3 antibody [EPR28430-76]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calponin 3 antibody [EPR28430-76] (AB314329)

Immunohistochemical analysis of paraffin-embedded human placenta tissue labeling Calponin 3 with ab314329 at 1/4000 (0.128 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on chorionic villi of human placenta (PMID : 20861310). The section was incubated with ab314329 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrumentIncubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. 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

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Immunocytochemistry/ Immunofluorescence - Anti-Calponin 3 antibody [EPR28430-76] (AB314329)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling Calponin 3 with ab314329 at 1/100 (5.1 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 HeLa cell line. Negative control : MCF7 (PMID : 30911294).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 (Red). 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-Calponin 3 antibody [EPR28430-76] (AB314329)

Immunohistochemical analysis of paraffin-embedded human skeletal muscle tissue labeling Calponin 3 with ab314329 at 1/4000 (0.128 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : no staining on human skeletal muscle. The section was incubated with ab314329 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrumentIncubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. 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.

• IP

Supplier Data

Immunoprecipitation - Anti-Calponin 3 antibody [EPR28430-76] (AB314329)

Calponin 3 was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab314329 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab314329 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 : ab314329 IP in HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab314329 in HeLa whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Lysate was freshly made and used for Western blotting immediately to minimize protein degradation.

All lanes:

Immunoprecipitation - Anti-Calponin 3 antibody [EPR28430-76] (ab314329) 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: 24s

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Calponin 3 antibody [EPR28430-76] (AB314329)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling Calponin 3 with ab314329 at 1/100 (5.1 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 NIH/3T3 cell line. 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 (Red). 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-Calponin 3 antibody [EPR28430-76] (AB314329)

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Calponin 3 with ab314329 at 1/4000 (0.128 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on mouse colon. The section was incubated with ab314329 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrumentIncubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. 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-Calponin 3 antibody [EPR28430-76] (AB314329)

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling Calponin 3 with ab314329 at 1/4000 (0.128 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on rat colon. The section was incubated with ab314329 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrumentIncubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. 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.

• IP

Supplier Data

Immunoprecipitation - Anti-Calponin 3 antibody [EPR28430-76] (AB314329)

Calponin 3 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab314329 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab314329 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate Lane 2 : ab314329 IP in NIH/3T3 (mouse embryonic fibroblast) whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab314329 in NIH/3T3 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Lysate was freshly made and used for Western blotting immediately to minimize protein degradation.

All lanes:

Immunoprecipitation - Anti-Calponin 3 antibody [EPR28430-76] (ab314329) at 1/30 dilution

All lanes:

NIH/3T3 (mouse embryonic fibroblast) 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-Calponin 3 antibody [EPR28430-76] (AB314329)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Lysates in lane 2-5 were freshly made and used for Western blotting immediately to minimize protein degradation. The identity of the bands around 25 kDa are unknown.

All lanes:

Western blot - Anti-Calponin 3 antibody [EPR28430-76] (ab314329) at 1/1000 dilution

Lane 1:

Human testis tissue lysate at 20 µg

Lane 2:

Mouse brain tissue lysate at 20 µg

Lane 3:

Mouse spleen tissue lysate at 20 µg

Lane 4:

Rat brain tissue lysate at 20 µg

Lane 5:

Rat testis tissue lysate at 20 µg

Secondary

Lanes 1 - 5:

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 1/2000 dilution

Lanes 1 - 5:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/100000 dilution

Observed band size: 40 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-Calponin 3 antibody [EPR28430-76] (AB314329)

Blocking and diluting buffer and concentration : 5% NFDM/TBST In lanes 5-7, lysates were stored at -80℃ prior to Western Blotting. In lanes 1-4, lysate were freshly made and used for Western Blotting immediately to minimize protein degradation, showing a reduction in the intensity of bands between 15 kDa and 30 kDa.

All lanes:

Western blot - Anti-Calponin 3 antibody [EPR28430-76] (ab314329) at 1/1000 dilution

Lanes 1 and 6:

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

Lanes 2 and 5:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lanes 3 and 7:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 4:

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: 40 kDa

false

Exposure time: 10s

• WB

Supplier Data

Western blot - Anti-Calponin 3 antibody [EPR28430-76] (AB314329)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Low expression : MCF7(PMID : 30911294). Lysates were freshly made and used for Western blotting immediately to minimize protein degradation. The identity of the bands around 25 kDa are unknown. In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Calponin 3 antibody [EPR28430-76] (ab314329) at 1/1000 dilution

Lane 1:

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

Lane 2:

MCF7 (human breast adenocarcinoma epithelial 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: 40 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-Calponin 3 antibody [EPR28430-76] (AB314329)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Negative control : Mouse skeletal muscle Lysates were freshly made and used for Western blotting immediately to minimize protein degradation. The identity of the bands around 25 kDa are unknown. In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Calponin 3 antibody [EPR28430-76] (ab314329) at 1/1000 dilution

Lane 1:

Mouse testis tissue lysate at 20 µg

Lane 2:

Mouse skeletal muscle tissue 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: 40 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-Calponin 3 antibody [EPR28430-76] (AB314329)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The recombinant proteins of Calponin 1, Calponin 2 and Calponin 3 were made in house. This antibody does not react with human Calponin 1 and Calponin 2. In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-Calponin 3 antibody [EPR28430-76] (ab314329) at 1/1000 dilution

Lane 1:

His-tagged human Calponin 2 recombinant protein at 20 ng

Lane 2:

His-tagged human Calponin 1 recombinant protein at 20 ng

Lane 3:

His-tagged human Calponin 3 recombinant fragment at 20 ng

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: 17 kDa

false

Exposure time: 15s