Anti-Connexin 43 / GJA1 抗体 [EPR21153] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Connexin 43 / GJA1 antibody [EPR21153] - BSA and Azide free (AB235625)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab217676).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Connexin 43 / GJA1 antibody [EPR21153] - BSA and Azide free (AB235625)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab217676).

• IP

Supplier Data

Immunoprecipitation - Anti-Connexin 43 / GJA1 antibody [EPR21153] - BSA and Azide free (AB235625)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab217676).

All lanes:

Immunoprecipitation - Anti-Connexin 43 / GJA1 antibody [EPR21153] - Intercellular Junction Marker (<a href='/products/primary-antibodies/connexin-43-gja1-antibody-epr21153-intercellular-junction-marker-ab217676'>ab217676</a>)

Predicted band size: 43 kDa

Observed band size: 26 kDa,43 kDa

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• WB

Unknown

Western blot - Anti-Connexin 43 / GJA1 antibody [EPR21153] - BSA and Azide free (AB235625)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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] - BSA and Azide free (ab235625) 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='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 43 kDa

Observed band size: 20-32 kDa

false

• WB

Unknown

Western blot - Anti-Connexin 43 / GJA1 antibody [EPR21153] - BSA and Azide free (AB235625)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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] - BSA and Azide free (ab235625) at 1/1000 dilution

Lane 1:

Wild-type HEK 293 whole cell lysate at 20 µg

Lane 2:

GJA1 knockout HEK 293 whole cell lysate at 20 µg

Lane 3:

Hela whole cell lysate at 20 µg

Predicted band size: 43 kDa

false

• WB

Lab

Western blot - Anti-Connexin 43 / GJA1 antibody [EPR21153] - BSA and Azide free (AB235625)

This data was developed using ab217676, the same antibody clone in a different buffer formulation.

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 (<a href='/products/primary-antibodies/connexin-43-gja1-antibody-epr21153-intercellular-junction-marker-ab217676'>ab217676</a>) 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='/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