重组Anti-Calcineurin A抗体[EPR24997-22]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calcineurin A antibody [EPR24997-22] (AB282104)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labelling Calcineurin A with ab282104 at 1/2000 dilution (0.267 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on human cerebrum (PMID : 28524178). The section was incubated with ab282104 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calcineurin A antibody [EPR24997-22] (AB282104)

Immunohistochemical analysis of paraffin-embedded Parental HAP1 cell pellet (A) and PPP3CA KO HAP1 cell pellet (B) labelling Calcineurin A with ab282104 at 1/100 dilution (5.33 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on parental HAP1 cell pellet (A) and no staining on PPP3CA KO HAP1 cell pellet (B). The section was incubated with ab282104 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 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-Calcineurin A antibody [EPR24997-22] (AB282104)

Immunohistochemical analysis of paraffin-embedded Human colon carcinoma tissue labelling Calcineurin A with ab282104 at 1/2000 dilution (0.267 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Cytoplamic staining on human colon carcinoma. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Calcineurin A antibody [EPR24997-22] (AB282104)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized HeLa (Human cervix adenocarcinoma) cells labelling Calcineurin A with ab282104 at 1/50 dilution (10.66 ug/ml), followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in HeLa cell line is observed.

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

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Calcineurin A antibody [EPR24997-22] (AB282104)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (Human cervix adenocarcinoma) cells labelling Calcineurin A with ab282104 at 1/500 dilution (0.1ug) (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). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calcineurin A antibody [EPR24997-22] (AB282104)

Immunohistochemical analysis of paraffin-embedded Human astrocytoma tissue labelling Calcineurin A with ab282104 at 1/2000 dilution (0.267 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Cytoplamic staining on human astrocytoma. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)

• IP

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Immunoprecipitation - Anti-Calcineurin A antibody [EPR24997-22] (AB282104)

Calcineurin A was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma) whole cell lysate with ab282104 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab282104 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (Human cervix adenocarcinoma) whole cell lysate 10 ug

Lane 2 : ab282104 IP in HeLa whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab282104 in HeLa whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 minutes

All lanes:

Immunoprecipitation - Anti-Calcineurin A antibody [EPR24997-22] (ab282104)

Predicted band size: 59 kDa

Observed band size: 32 kDa,59 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calcineurin A antibody [EPR24997-22] (AB282104)

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labelling Calcineurin A with ab282104 at 1/2000 dilution (0.267 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on rat cerebrum. The section was incubated with ab282104 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 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-Calcineurin A antibody [EPR24997-22] (AB282104)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labelling Calcineurin A with ab282104 at 1/2000 dilution (0.267 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on mouse cerebrum. The section was incubated with ab282104 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Calcineurin A antibody [EPR24997-22] (AB282104)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling Calcineurin A with ab282104 at 1/500 dilution (0.1ug) (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). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Calcineurin A antibody [EPR24997-22] (AB282104)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling Calcineurin A with ab282104 at 1/50 dilution (10.66 ug/ml), followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in NIH/3T3 cell line is observed.

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

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-Calcineurin A antibody [EPR24997-22] (AB282104)

Calcineurin A was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab282104 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab282104 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 lysate10 ug

Lane 2 : ab282104 IP in NIH/3T3 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab282104 in NIH/3T3 whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 59 seconds

All lanes:

Immunoprecipitation - Anti-Calcineurin A antibody [EPR24997-22] (ab282104)

Predicted band size: 59 kDa

Observed band size: 32 kDa,59 kDa

false

• WB

Lab

Western blot - Anti-Calcineurin A antibody [EPR24997-22] (AB282104)

Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

Lanes 1-4 : Merged signal (red and green). Green - ab282104 observed at 59/32 kDa. Red - loading control ab8245 observed at 36 kDa.

ab282104 Anti-PPP3CA antibody [EPR24997-22 HT] was shown to specifically react with PPP3CA in wild-type HAP1 cells. Loss of signal was observed when the knockout cell line was used. Wild-type and PPP3CA knockout samples were subjected to SDS-PAGE. ab282104 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4℃ overnight at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

The expression pattern & observed MW are consistent with what has been described in the literature (PMID : 17785681).

All lanes:

Western blot - Anti-Calcineurin A antibody [EPR24997-22] (ab282104) at 1/1000 dilution

Lane 1:

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

Lane 2:

PPP3CA knockout HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 59 kDa

Observed band size: 32 kDa,59 kDa

false

• WB

Lab

Western blot - Anti-Calcineurin A antibody [EPR24997-22] (AB282104)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

The expression pattern & observed MW are consistent with what has been described in the literature (PMID : 17785681).

Exposure time : Lane 1-2 : 3.25 seconds Lane 3 : 26 seconds Lane 4 : 59 seconds

All lanes:

Western blot - Anti-Calcineurin A antibody [EPR24997-22] (ab282104) 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 lung tissue lysate at 20 µg

Lane 4:

Rat lung tissue lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 59 kDa

Observed band size: 32 kDa,59 kDa

false

• WB

Lab

Western blot - Anti-Calcineurin A antibody [EPR24997-22] (AB282104)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

This antibody does not recognize human PPP3CB and PPP3CC. These rec proteins are made in-house and expressed from E.coli expression systems.

Exposure time : 15 seconds

All lanes:

Western blot - Anti-Calcineurin A antibody [EPR24997-22] (ab282104) at 1/1000 dilution

Lane 1:

Human PPP3CA recombinant protein at 0.01 µg

Lane 2:

Human PPP3CB recombinant protein at 0.01 µg

Lane 3:

Human PPP3CC recombinant protein at 0.01 µg

Secondary

All lanes:

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

Predicted band size: 59 kDa

Observed band size: 32 kDa,59 kDa

false