All lanes : Anti-Connexin 43 / GJA1 antibody [EPR21153] (ab217676) at 1/1000 dilution

Lane 1 : Wild-type HEK 293 whole cell lysate
Lane 2 : GJA1 knockout HEK 293 whole cell lysate
Lane 3 : Hela whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 43 kDa

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 : Anti-Connexin 43 / GJA1 antibody [EPR21153] (ab217676) at 1/1000 dilution

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : Human testis tissue lysate
Lane 3 : Human fetal brain tissue lysate
Lane 4 : Human breast cancer tissue lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 43 kDa
Observed band size: 26-32 kDa why is the actual band size different from the predicted?

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).

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).

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).

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).