Anti-Connexin 43 / GJA1 (phospho Y265) antibody [EPR26416-134] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Connexin 43 / GJA1 (phospho Y265) antibody [EPR26416-134] - BSA and Azide free (AB312837)

This data was developed using ab312836, the same antibody clone in a different buffer formulation. Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling Connexin 43 / GJA1 (phospho Y265) with ab312836 at 1/8000 (0.061 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 and membranous staining in HeLa cells transfected with a human Connexin-43 wildtype and V-SRC wildtype expression vectors containing a myc tag and showing no staining in HeLa cells transfected with a human Connexin-43 Y265F and V-SRC wildtype expression vectors containing a myc tag. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). Myc-Tag Mouse mAb (Alexa Fluor® 647) was used to counterstain tubulin at 1/100 (0.38 ug/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.

• IP

Supplier Data

Immunoprecipitation - Anti-Connexin 43 / GJA1 (phospho Y265) antibody [EPR26416-134] - BSA and Azide free (AB312837)

This data was developed using ab312836, the same antibody clone in a different buffer formulation. Connexin 43 / GJA1 (phospho Y265) was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) cells co-transfected with a human GJA1 expression vector containing a myc-His-tag® and a RSV-SR-A V-SRC expression vector containing a myc-His-tag® whole cell lysate with ab312836 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab312836 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) cells co-transfected with a human GJA1 expression vector containing a myc-His-tag® and a RSV-SR-A V-SRC expression vector containing a myc-His-tag® whole cell lysate Lane 2 : HeLa (human cervical adenocarcinoma epithelial cell) cells co-transfected with a human GJA1 expression vector containing a myc-His-tag® and a RSV-SR-A V-SRC expression vector containing a myc-His-tag® whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab312836 in HeLa cells co-transfected with a human GJA1 expression vector containing a myc-His-tag® and a RSV-SR-A V-SRC expression vector containing a myc-His-tag® whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 8 seconds

All lanes:

Immunoprecipitation - 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 1/30 dilution

All lanes:

HeLa cells co-transfected with a human GJA1 expression vector containing a myc-His-tag® and a RSV-SR-A V-SRC expression vector containing a myc-His-tag® whole cell lysate at 2 µg

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: 8s

• WB

Supplier Data

Western blot - Anti-Connexin 43 / GJA1 (phospho Y265) antibody [EPR26416-134] - BSA and Azide free (AB312837)

This data was developed using ab312836, the same antibody clone in a different buffer formulation.

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

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-GAPDH antibody (ab181602) loading control staining at 1/200000 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:

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

Lane 2:

HeLa treated with 30mM pervanadate for 30 minutes 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: 180s

• WB

Lab

Western blot - Anti-Connexin 43 / GJA1 (phospho Y265) antibody [EPR26416-134] - BSA and Azide free (AB312837)

This data was developed using ab312836, the same antibody clone in a different buffer formulation.

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 : 15371442，PMID : 30054380).

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:

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

Lane 2:

HeLa treated with 1mM pervanadate for 60 minutes 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 1/1000 dilution

Observed band size: 43 kDa

false

Exposure time: 60s

• WB

Supplier Data

Western blot - Anti-Connexin 43 / GJA1 (phospho Y265) antibody [EPR26416-134] - BSA and Azide free (AB312837)

This data was developed using ab312836, the same antibody clone in a different buffer formulation. 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). 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] (ab235585) total protein control staining at 1/1000 dilution. Exposure time : 70 seconds

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

Lane 3:

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

Lane 4:

HeLa cells co-transfected with a mouse 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

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: 48 kDa,55 kDa

false

Exposure time: 70s

• WB

Supplier Data

Western blot - Anti-Connexin 43 / GJA1 (phospho Y265) antibody [EPR26416-134] - BSA and Azide free (AB312837)

This data was developed using ab312836, the same antibody clone in a different buffer formulation.

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

• Dot

Supplier Data

Dot Blot - Anti-Connexin 43 / GJA1 (phospho Y265) antibody [EPR26416-134] - BSA and Azide free (AB312837)

This data was developed using ab312836, the same antibody clone in a different buffer formulation. Dot blot analysis of Connexin 43 / GJA1 (phospho Y265) using ab312836 at 1 : 1000 (0.484 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution. Lane 1 : Connexin 43 / GJA1 (phospho Y265) peptide a Lane 2 : Connexin 43 / GJA1 (phospho Y265) peptide b Lane 3 : Connexin 43 / GJA1 non-phospho peptide Exposure time : 180 seconds Blocking and diluting buffer and concentration : 5% NFDM/TBST