All lanes : Anti-Pepsinogen II/PGC antibody [EPR22843-269] (ab255826) at 1/1000 dilution

Lane 1 : Human stomach lysate
Lane 2 : Human testis lysate
Lane 3 : MDA-MB-231 (human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 4 : Rat stomach lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1-3 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution
Lane 4 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 42 kDa
Observed band size: 32,38 kDa why is the actual band size different from the predicted?

Bands of 38KDa and 32KDa represent the precursor and the mature form.  The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 28424202). 

Negative control: Human testis and MDA-MB-231 (PMID: 11259249 and 8663058 ).

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

Exposure Time: Lane 1:10 seconds; Lanes 2-4:3 minutes.

Anti-Pepsinogen II/PGC antibody [EPR22843-269] (ab255826) at 1/1000 dilution + Mouse stomach lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 42 kDa
Observed band size: 32,38 kDa why is the actual band size different from the predicted?

This blot was developed using a higher sensitivity ECL substrate. Blocking/Diluting buffer and concentration: 5% NFDM/TBST  Exposure Time: 37 seconds

Anti-Pepsinogen II/PGC antibody [EPR22843-269] (ab255826) at 1/1000 dilution + His-tagged Recombinant Human Pepsinogen C/PGC protein(aa1-388), 10ng

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

Predicted band size: 42 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?

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

Exposure Time: 3 minutes.

Immunohistochemical analysis of paraffin-embedded Rat stomach tissue labeling Pepsinogen II/PGC with ab255826 at 1/5000 dilution (0.129 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat stomach. The section was incubated with ab255826 for 10 mins at RT. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control/ Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Mouse stomach tissue labeling Pepsinogen II/PGC with ab255826 at 1/5000 dilution (0.129 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse stomach. The section was incubated with ab255826 for 10 mins at RT. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control/ Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Human stomach tissue labeling Pepsinogen II/PGC with ab255826 at 1/5000 dilution (0.129 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human stomach (PMID 10883891). The section was incubated with ab255826 for 10 mins at RT. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control/ Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunohistochemical analysis of frozen section of 4% PFA-fixed, 0.2% Triton X-100 permeabilized rat stomach tissue labeling Annexin-4/ANXA4 with ab255826 at 1/500 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (green). Positive staining on rat gastric mucosa (PMID: 10883891) is observed. The nuclear counter stain is DAPI (blue).
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is 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).

Immunohistochemical analysis of frozen section of 4% PFA-fixed, 0.2% Triton X-100 permeabilized mouse stomach tissue labeling Annexin-4/ANXA4 with ab255826 at 1/500 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (green). Positive staining on mouse gastric mucosa (PMID: 10883891) is observed. The nuclear counter stain is DAPI (blue).
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is 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).