Anti-Connexin 43 / GJA1 antibody [EPR21153] - Intercellular Junction Marker

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Connexin 43 / GJA1 antibody [EPR21153] - Intercellular Junction Marker (AB217676)

Immunohistochemical analysis of paraffin-embedded human cardiac muscle tissue labeling Connexin 43 / GJA1 with ab217676 at 1/500 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) ready to use. Positive staining on human cardiac muscle (PMID : 25018732) is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) ready to use.

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Connexin 43 / GJA1 antibody [EPR21153] - Intercellular Junction Marker (AB217676)

Immunohistochemical analysis of paraffin-embedded human prostate tissue labeling Connexin 43 / GJA1 with ab217676 at 1/500 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) ready to use. Positive staining on basal cells of human prostate gland (PMID : 20735413) is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) ready to use.

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

• IP

Supplier Data

Immunoprecipitation - Anti-Connexin 43 / GJA1 antibody [EPR21153] - Intercellular Junction Marker (AB217676)

Connexin 43 / GJA1 was immunoprecipitated from 0.35 mg of human testis lysate with ab217676 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab217676 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1 : Human testis lysate 10 μg (Input).
Lane 2 : ab217676 IP in human testis lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab217676 in human testis lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.
Exposure time : 1 minute.

The expression profile is consistent with the existence of natural variants described in the literature (PMID : 24210816; PMID : 28576298).

All lanes:

Immunoprecipitation - Anti-Connexin 43 / GJA1 antibody [EPR21153] - Intercellular Junction Marker (ab217676)

Predicted band size: 43 kDa

Observed band size: 26 kDa,43 kDa

false

• WB

Lab

Western blot - Anti-Connexin 43 / GJA1 antibody [EPR21153] - Intercellular Junction Marker (AB217676)

Lanes 1 - 4 : Merged signal (red and green). Green - ab217676 observed at 43 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab217676 was shown to recognize Connexin 43 / GJA1 in wild-type HEK 293 cells as signal was lost at the expected MW in GJA1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and GJA1 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. ab217676 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Connexin 43 / GJA1 antibody [EPR21153] - Intercellular Junction Marker (ab217676) at 1/1000 dilution

Lane 1:

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

Lane 2:

GJA1 knockout HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

Western blot - Human GJA1 knockout HEK-293 cell line (<a href='/en-us/products/cell-lines/human-gja1-knockout-hek-293-cell-line-ab261719'>ab261719</a>)

Lane 3:

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

Predicted band size: 43 kDa

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• WB

Supplier Data

Western blot - Anti-Connexin 43 / GJA1 antibody [EPR21153] - Intercellular Junction Marker (AB217676)

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure times.
Lane 1 : 37 seconds.
Lane 2 : 15 seconds.
Lane 3 : 103 seconds.
Lane 4 : 3 minutes.

The expression profile is consistent with the existence of natural variants described in the literature (PMID : 24210816; PMID : 28576298).

All lanes:

Western blot - Anti-Connexin 43 / GJA1 antibody [EPR21153] - Intercellular Junction Marker (ab217676) at 1/1000 dilution

Lane 1:

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

Lane 2:

Human testis tissue lysate at 20 µg

Lane 3:

Human fetal brain tissue lysate at 20 µg

Lane 4:

Human breast cancer 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

Predicted band size: 43 kDa

Observed band size: 26-32 kDa

false

• WB

Lab

Western blot - Anti-Connexin 43 / GJA1 antibody [EPR21153] - Intercellular Junction Marker (AB217676)

Western blot : Anti-GJA1 antibody [EPR21153] (ab217676) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab217676 was shown to bind specifically to GJA1. A band was observed at 45 kDa in wild-type U-87 MG cell lysates with no signal observed at this size in GJA1 knockout cell line. To generate this image, wild-type and GJA1 knockout U-87 MG cell lysates were analysed. First, 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 IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Connexin 43 / GJA1 antibody [EPR21153] - Intercellular Junction Marker (ab217676) at 1/1000 dilution

Lane 1:

Wild-type U-87 MG cell lysate at 20 µg

Lane 2:

Western blot - Human GJA1 knockout U-87 MG cell line (ab306676)

Lane 3:

Wild-type HEK-293 cell lysate at 20 µg

Lane 4:

GJA1 knockout HEK-293 <a href='/en-us/products/cell-lysates/human-gja1-knockout-hek-293-cell-lysate-ab261658'>ab261658</a> cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 45 kDa

false

• WB

Supplier Data

Western blot - Anti-Connexin 43 / GJA1 antibody [EPR21153] - Intercellular Junction Marker (AB217676)

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 18649180; PMID : 30448479).

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

Alkaline phosphatase. - : untreated membrane; + : phosphatase treated membrane.

In Western blot, anti-His antibody (ab213204) staining at 1/5000 dilution.

Anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

Anti-Connexin 43 / GJA1 antibody [EPR21153] (ab217676) total protein control staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Connexin 43 / GJA1 (phospho Y265) antibody [EPR26416-134] (<a href='/en-us/products/primary-antibodies/connexin-43-gja1-phospho-y265-antibody-epr26416-134-ab312836'>ab312836</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) cells transfected with an empty vector containi a myc-His-tag® whole cell lysate at 20 µg

Lane 2:

HeLa cells transfected with a human GJA1 expression vector containi a myc-His-tag® whole cell lysate at 20 µg

Lane 3:

HeLa cells transfected with a human GJA1(Mutant Y265F) expression vector containi a myc-His-tag® whole cell lysate at 20 µg

Lane 4:

HeLa cells transfected with a RSV-SR-A V-SRC expression vector containi a myc-His-tag® whole cell lysate at 20 µg

Lane 5:

HeLa cells co-transfected with a human GJA1 expression vector containi a myc-His-tag® and a RSV-SR-A V-SRC expression vector containi a myc-His-tag® whole cell lysate at 20 µg

Lane 6:

HeLa cells co-transfected with a human GJA1(Mutant Y265F) expression vector containi a myc-His-tag® and a RSV-SR-A V-SRC expression vector containi a myc-His-tag® 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: 43 kDa

true

Exposure time: 92s