Anti-RCN1/RCN antibody [EPR17163-117]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-RCN1/RCN antibody [EPR17163-117] (AB210404)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed SH-SY5Y (Human neuroblastoma cell line from bone marrow) cells labeling RCN1/RCNwith ab210404 at 1/700 dilution (red) compared with a Rabbit IgG,monoclonal[EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (Alexa Fluor^® 488) at 1/500 dilution was used as the secondary antibody.

Cells were permeabilised with 90% methanol (diluted with 1X PBS).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RCN1/RCN antibody [EPR17163-117] (AB210404)

Immunohistochemical analysis of paraffin-embedded Human lung adenocarcinoma tissue labeling RCN1/RCN with ab210404 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on tumor cells of Human lung adenocarcinoma is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RCN1/RCN antibody [EPR17163-117] (AB210404)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling RCN1/RCN with ab210404 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on neurons of Human cerebrum is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-RCN1/RCN antibody [EPR17163-117] (AB210404)

Immunofluorescent analysis of 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilized SH-SY5Y (Human neuroblastoma cell line from bone marrow) cells labeling RCN1/RCN with ab210404 at 1/500 dilution followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on SH-SY5Y cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows : -

-ve control 1 : ab210404 at 1/500 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2 : ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

• IP

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Immunoprecipitation - Anti-RCN1/RCN antibody [EPR17163-117] (AB210404)

RCN1/RCN was immunoprecipitated from 1mg of 293T (Human epithelial cell line from embryonic kidney) whole cell lysate with ab210404 at 1/50 dilution.

Western blot was performed from the immunoprecipitate using ab210404 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : 293T whole cell lysate, 10μg (Input).

Lane 2 : ab210404 IP in 293T whole cell lysate.

Lane 3 : Rabbit IgG,monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab210404 in 293T whole cell lysate.

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

Exposure time : 1 second.

All lanes:

Immunoprecipitation - Anti-RCN1/RCN antibody [EPR17163-117] (ab210404)

Predicted band size: 39 kDa

false

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-RCN1/RCN antibody [EPR17163-117] (AB210404)

Immunofluorescent analysis of 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilized Neuro-2a (Mouse neuroblastoma cell line) cells labeling RCN1/RCN with ab210404 at 1/500 dilution followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on Neuro-2a cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows : -

-ve control 1 : ab210404 at 1/500 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2 : ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-RCN1/RCN antibody [EPR17163-117] (AB210404)

Immunofluorescent analysis of 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling RCN1/RCN with ab210404 at 1/500 dilution followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on NIH/3T3 cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows : -

-ve control 1 : ab210404 at 1/500 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2 : ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RCN1/RCN antibody [EPR17163-117] (AB210404)

Immunohistochemical analysis of paraffin-embedded Mouse cerebral cortex tissue labeling RCN1/RCN with ab210404 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on neurons of mouse cerebral cortex is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RCN1/RCN antibody [EPR17163-117] (AB210404)

Immunohistochemical analysis of paraffin-embedded Rat hippocampus tissue labeling RCN1/RCN with ab210404 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on neurons of rat hippocampus is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-RCN1/RCN antibody [EPR17163-117] (AB210404)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling RCN1/RCNwith ab210404 at 1/700 dilution (red) compared with a Rabbit IgG,monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (Alexa Fluor^® 488) at 1/500 dilution was used as the secondary antibody.

Cells were permeabilised with 90% methanol (diluted with 1X PBS).

• WB

Lab

Western blot - Anti-RCN1/RCN antibody [EPR17163-117] (AB210404)

Lanes 1- 4 : Merged signal (red and green). Green - ab210404 observed at 45 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

ab210404 was shown to react with RCN1 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266098 (knockout cell lysate ab258172) was used. Wild-type HEK-293T and RCN1 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab210404 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 2000 dilution and a 1 in 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-RCN1/RCN antibody [EPR17163-117] (ab210404) at 1/2000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

RCN1 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human RCN1 (RCN) knockout HEK-293T cell line (<a href='/ko/products/cell-lines/human-rcn1-rcn-knockout-hek-293t-cell-line-ab266098'>ab266098</a>)

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Daudi cell lysate at 20 µg

Predicted band size: 39 kDa

Observed band size: 45 kDa

false

• WB

Supplier Data

Western blot - Anti-RCN1/RCN antibody [EPR17163-117] (AB210404)

Blocking/Dilution buffer : 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID : 8416973).

All lanes:

Western blot - Anti-RCN1/RCN antibody [EPR17163-117] (ab210404) at 1/5000 dilution

Lane 1:

Human placenta lysate at 20 µg

Lane 2:

Human fetal kidney lysate at 20 µg

Lane 3:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 4:

SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate at 20 µg

Lane 5:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 6:

E14 mouse embryo lysate at 20 µg

Lane 7:

E14 rat embryo lysate at 20 µg

Lane 8:

Neuro-2a (Mouse neuroblastoma cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/100000 dilution

Predicted band size: 39 kDa

Observed band size: 44 kDa,46 kDa

false

Exposure time: 30s

• WB

Lab

Western blot - Anti-RCN1/RCN antibody [EPR17163-117] (AB210404)

Western blot : Anti-RCN1/RCN antibody [EPR17163-117] ab210404 staining at 1/2000 dilution, shown in green; Mouse anti-CANX ab238078 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 45 kDa in Wild-type HEK-293T cell lysates with no signal observed at this size in RCN1 knockout HEK-293T cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-RCN1/RCN antibody [EPR17163-117] (ab210404) at 1/2000 dilution

Lane 1:

Wild-type HEK-293T cell lysates at 20 µg

Lane 2:

Western blot - Human RCN1 (RCN) knockout HEK-293T cell line (<a href='/ko/products/cell-lines/human-rcn1-rcn-knockout-hek-293t-cell-line-ab266098'>ab266098</a>) at 20 µg

Lane 3:

HeLa cell lysates at 20 µg

Lane 4:

Daudi cell lysates at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 45 kDa

Observed band size: 45 kDa

false

• WB

Supplier Data

Western blot - Anti-RCN1/RCN antibody [EPR17163-117] (AB210404)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1-8 : 1 minute; Lane 9,10 and 11 : 10 seconds.

All lanes:

Western blot - Anti-RCN1/RCN antibody [EPR17163-117] (ab210404) at 1/2000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Mouse heart lysate at 10 µg

Lane 3:

Mouse kidney lysate at 10 µg

Lane 4:

Mouse spleen lysate at 10 µg

Lane 5:

Rat brain lysate at 10 µg

Lane 6:

Rat heart lysate at 10 µg

Lane 7:

Rat kidney lysate at 10 µg

Lane 8:

Rat spleen lysate at 10 µg

Lane 9:

C6 (Rat glial tumor cell line) whole cell lysate at 10 µg

Lane 10:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Lane 11:

NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/100000 dilution

Predicted band size: 39 kDa

Observed band size: 44 kDa,46 kDa

false