Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid carcinoma tissue labelling GSK3 (alpha + beta) with purified ab68476 at 1/2000. Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes. ab209101, a Rabbit specific IHC polymer detection kit HRP/DAB was used. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Positive staining on human thyroid carcinoma without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B).

The section was incubated with ab68476 for 30 minutes at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument.

All lanes : Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476) at 1/1000 dilution (purified)

Lane 1 : Zebrafish lysates
Then the membrane was incubated with phosphatase
Lane 2 : Untreated Zebrafish lysates

Lysates/proteins at 15 µg per lane.

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

Predicted band size: 47-52 kDa
Observed band size: 47-52 kDa

Blocking and diluting buffer: 5% NFDM/TBST

 

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovarian carcinoma tissue labelling GSK3 (alpha + beta) with purified ab68476 at 1/2000. Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes. ab209101, a Rabbit specific IHC polymer detection kit HRP/DAB was used. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Positive staining on human ovarian carcinoma without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B).

The section was incubated with ab68476 for 30 minutes at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human glioma tissue sections labeling GSK3 (alpha + beta) with Purified ab68476 at 1:50 dilution (5.3 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

All lanes : Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476) at 1/1000 dilution (purified)

Lane 1 : Mouse brain lysates.
Then the membrane was incubated with phosphatase
Lane 2 : Untreated Mouse brain lysates

Lysates/proteins at 15 µg per lane.

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

Predicted band size: 47-52 kDa

Blocking and diluting buffer: 5% NFDM/TBST

All lanes : Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476) at 1/500 dilution (unpurified)

Lane 1 : SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysates
Lane 2 : SH-SY5Y (Human neuroblastoma epithelial cell) treated with nerve growth factor at 100ng/ml for 5 minutes. Whole cell lysates.
Lane 3 : SH-SY5Y (Human neuroblastoma epithelial cell) treated with nerve growth factor at 100ng/ml for 5 minutes. Whole cell lysates. Then the membrane was incubated with phosphatase.

Lysates/proteins at 15 µg per lane.

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

Predicted band size: 47-52 kDa
Observed band size: 47-52 kDa


Exposure time: 10 seconds

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

Human brain tissue stained with unpurified ab68476 at 1/100 dilution.

Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

GSK3 (alpha + beta) (phospho Y216 + Y279) was immunoprecipitated from 10μg HeLa (human cervix adenocarcinoma) whole cell lysate with ab68476 at 1/50 dilution (2μg in 0.35mg lysates). Western blot was performed from the immunoprecipitate using ab68476 at 1/200 dilution (9 μg/ml). VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1 (Input): HeLa (human cervix adenocarcinoma) treated with 1uM staurosporine for 4h whole cell lysate 10μg
Lane 2 (+): HeLa (human cervix adenocarcinoma) treated with 1uM staurosporine for 4h whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab68476 in HeLa (human cervix adenocarcinoma) treated with 1uM staurosporine for 4h whole cell lysate

Exposure Time: 30 seconds
Blocking and diluting buffer and concentration: 5% NFDM/TBST.

 

All lanes : Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476) (unpurified)

Lane 1 : PC12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 2 : Whole cell lysate from PC12 (Rat adrenal gland pheochromocytoma cell line) cells treated with nerve growth factor-ß at 100ng/ml for 5 minutes.
Lane 3 : Whole cell lysate from PC12 (Rat adrenal gland pheochromocytoma cell line) cells ) treated with nerve growth factor-ß at 100ng/ml for 5 minutes. Membrane incubated with phosphatase

Lysates/proteins at 15 µg per lane.

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

Developed using the ECL technique.

Predicted band size: 47-52 kDa


Exposure time: 3 seconds

Blocking and dilution buffer: 5% NFDM/TBST

Dot Blot analysis of Lane 1: Human GSK3 (alpha + beta) (pY216 + pY279) phospho peptide and Lane 2: Human GSK3 (alpha + beta) non-phospho peptide labeling GSK3 (alpha + beta) (phospho Y216 + Y279) with ab68476 at 1/1000 dilution. 5% NFDM/TBST was used as the diluting and blocking buffer. ab97051 Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated was used as the secondary antibody at 1/100000 dilution. Exposure time: 3 minutes.

All lanes : Anti-GSK3 (alpha + beta) (phospho Y216 + Y279) antibody [EPR933Y] (ab68476) at 1/2000 dilution (unpurified)

Lane 1 : HEK-293 (Human epithelial cell line from embryonic kidney) cell lysate, cells untreated.
Lane 2 : HEK-293 cell lysate, cells treated with AP.

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP conjugated Goat anti-rabbit at 1/2000 dilution

Predicted band size: 47-52 kDa
Observed band size: 47-52 kDa