All lanes : Anti-Connexin 43 / GJA1 antibody [EPR22955-101] (ab235585) at 1/1000 dilution

Lane 1 : Huh7( Human hepatocellular carcinoma epithelial cell)whole cell lysate
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : C2C12 (Mouse myoblasts myoblast)whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/20000 dilution

Exposure time: 180 seconds

ab235585 has low affinity in cell line samples. 

Blocking and dilution buffer: 5% NFDM/TBST

All lanes : Anti-Connexin 43 / GJA1 antibody [EPR22955-101] (ab235585) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate at 20 µg
Lane 2 : Mouse heart tissue lysate at 20 µg
Lane 3 : Rat brain tissue lysate at 10 µg
Lane 4 : Human brain tissue lysate at 10 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 43 kDa why is the actual band size different from the predicted?

Blocking and Dilution Buffer and concentration: 5% NFDM/TBST. The expression profile is consistent with the existence of natural variants described in the literature (PMID: 24210816, 28576298). Exposure times: Lane 1: 59 seconds; Lane 2: 15 seconds; Lane 3: 15 seconds; Lane 4: 26 seconds.

Connexin 43 / GJA1 was immunoprecipitated from 0.35 mg mouse brain lysate 10µg with ab235585 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab235585 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: Mouse brain lysate 10µg.

Lane 2: ab235585 IP in mouse brain lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab235585 in mouse brain lysate.

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

Exposure time: 10 seconds.

High MW band between 75kDa and 100kDa represents Connexin 43 dimers which described in literature. (PMID: 19460776).

 

Immunohistochemical analysis of paraffin-embedded rat cardiac muscle tissue labeling Connexin 43 / GJA1 with ab235585 at 1/1000 dilution (0.507 µg/ml) followed by a ready to use Goat Anti-Rabbit & Mouse IgG (HRP). Positive staining on rat myocardial intercalated disc is observed. The section was incubated with ab235585 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 Goat Anti-Rabbit & Mouse IgG (HRP).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling Connexin 43 / GJA1 with ab235585 at 1/1000 dilution (0.507 µg/ml) followed by a ready to use Goat Anti-Rabbit & Mouse IgG (HRP). Membranous staining on mouse testis is observed. The section was incubated with ab235585 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 Goat Anti-Rabbit & Mouse IgG (HRP).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded mouse cardiac muscle tissue labeling Connexin 43 / GJA1 with ab235585 at 1/1000 dilution (0.507 µg/ml) followed by a ready to use Goat Anti-Rabbit & Mouse IgG (HRP). Positive staining on mouse myocardial intercalated disc (PMID: 30228305) is observed. The section was incubated with ab235585 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 Goat Anti-Rabbit & Mouse IgG (HRP).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human cardiac muscle tissue labeling Connexin 43 / GJA1 with ab235585 at 1/500 dilution (1.01 µg/ml) followed by a ready to use Goat Anti-Rabbit & Mouse IgG (HRP). Positive staining on human myocardial intercalated disc is observed. The section was incubated with ab235585 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 Goat Anti-Rabbit & Mouse IgG (HRP).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen section of rat cardiac muscle tissue labeling Connexin 43 / GJA1 using ab235585 at 1/250 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (green). The nuclear counterstain is DAPI (blue). Positive staining on intercalated discs of rat cardiac muscle (PMID: 18678643) is observed.

Secondary antibody only control: Used PBS instead of primary antibody, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

 

4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen section of mouse cardiac muscle tissue labeling Connexin 43 / GJA1 using ab235585 at 1/250 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (green). The nuclear counterstain is DAPI (blue). Positive staining on intercalated discs of mouse cardiac muscle (PMID: 18678643) is observed.

Secondary antibody only control: Used PBS instead of primary antibody, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).