All lanes : Anti-Stanniocalcin 2/STC-2 antibody [EPR22866-10] (ab255610) at 1/1000 dilution

Lane 1 : HCT116 (human colorectal carcinoma epithelial cell), whole cell lysate
Lane 2 : 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: 33 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST The expression profile observed is consistent with what has been described in the literature (PMID:15367391, 25463045, 26361149). 

Exposure time: Lane 1: 3 minutes; Lane 2: 48 seconds.

All lanes : Anti-Stanniocalcin 2/STC-2 antibody [EPR22866-10] (ab255610) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : HepG2 (human hepatocellular carcinoma epithelial cell), whole cell lysate
Lane 3 : MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate
Lane 4 : HT-29 (human colorectal adenocarcinoma epithelial cell), whole cell lysate
Lane 5 : 293T (human embryonic kidney epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1-2 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051)
Lanes 3-5 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 33 kDa
Observed band size: 33/35 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST The expression profile observed is consistent with what has been described in the literature, the dual bands caused by different posttranslational modifications (PMID: 15367391, 25463045, 26361149). 

Exposure time: Lane 1-2: 48 seconds; Lane 3: 26 seconds; Lane 4: 114 seconds; Lane 5: 15 seconds.

STC2 was immunoprecipitated from 0.35 mg T-47D (human ductal breast epithelial tumor epithelial cell) whole cell lysate with ab255610 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab255610 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: T-47D (human ductal breast epithelial tumor epithelial cell) whole cell lysate 10ug

Lane 2: ab255610 IP in T-47D whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab255610 in T-47D whole cell lysate

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

Exposure time: 30 seconds.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labelling STC2 with ab255610 at 1/50 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized T-47D (human ductal breast epithelial tumor epithelial cell) cells labelling STC2 with ab255610 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

 

Immunohistochemical analysis of paraffin-embedded Human breast cancer (A) and its adjacent noncancerous breast tissue (B) tissue labeling STC2 with ab255610 at 1/ 200 dilution (2.29ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Strong cytoplasmic staining in human breast cancer tissue (A) while weak staining in its adjacent noncancerous breast tissue (B) (PMID: 18492817) is observed. The section was incubated with ab255610 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

Immunohistochemical analysis of paraffin-embedded Human lung cancer (A) and its adjacent noncancerous lung tissue (B) tissue labeling STC2 with ab255610 at 1/ 200 dilution (2.29ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Strong cytoplasmic staining in human lung cancer tissue (A) while weak staining in its adjacent noncancerous lung tissue (B) (PMID: 25463045) is observed. The section was incubated with ab255610 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labelling STC2 with ab255610 at 1/50 (9 ug/ml) dilution, followed by ab150077 AlexaFluor^® 488 Goat anti-Rabbit secondary antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing cytoplasmic staining in HepG2 cell line. Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor^® 488 Goat anti-Rabbit secondary at 1/1000 (2 ug/ml) dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized T-47D (human ductal breast epithelial tumor epithelial cell) cells labelling STC2 with ab255610 at 1/50 (9 ug/ml) dilution, followed by ab150077 AlexaFluor^® 488 Goat anti-Rabbit secondary antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing cytoplasmic staining in T-47D cell line. Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200  dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is Ab255610 anti-STC2 ab150077 AlexaFluor^® 488 Goat anti-Rabbit secondary at 1/1000 (2 ug/ml) dilution.