Phospho-GSK3 (alpha + beta) (Y216 + Y279) Recombinant Rabbit Monoclonal Antibody [SY26-05]
cat.: ET1607-54
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human, Mouse, Rat
Applications: WB, IF-Cell, IHC-P, IP, IF-Tissue, FC
Clonality: Monoclonal
Clone number: SY26-05
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 1ug/ul
Purification: Protein A affinity purified.
Molecular weight: Predicted band size: 51 kDa
Isotype: IgG
Immunogen: Synthetic phospho-peptide corresponding to residues surrounding Tyr216 and 279 of human GSK3(alpha+beta).
Positive control: 293T cell lysate, SH-SY5Y cell lysate, HeLa cell lysate, HepG2 cell lysate, MCF7 cell lysate, NIH/3T3 cell lysate, PC-12 cell lysate, mouse brain tissue lysate, rat brain tissue lysate, mouse cerebellum tissue lysates, A431, Hela, MCF-7, rat brain tissue, rat hippocampus tissue, mouse brain tissue, human thyroid carcinoma tissue.
Subcellular location: Cytoplasm, Nucleus, Cell membrane.
Recommended Dilutions:
  WB
  IF-Cell
  IHC-P
  IP
  IF-Tissue
  FC
1:500-1:1,000
1:100-1:500
1:200-1:800
Use at an assay dependent concentration.
1:200
1:1,000
Uniprot #: SwissProt: P49840 Human | P49841 Human | Q2NL51 Mouse | Q9WV60 Mouse | P18265 Rat | P18266 Rat
Alternative names: Factor A Glycogen synthase kinase 3 alpha Glycogen synthase kinase 3 beta GSK3 alpha GSK3 beta GSK3B
Images
ET1607-54_1.jpg Fig1: Western blot analysis of Phospho-GSK3 (alpha + beta) (Y216 + Y279) on different lysates with Rabbit anti-Phospho-GSK3 (alpha + beta) (Y216 + Y279) antibody (ET1607-54) at 1/5,000 dilution.

Lane 1: 293T cell lysate
Lane 2: SH-SY5Y cell lysate
Lane 3: HeLa cell lysate
Lane 4: HepG2 cell lysate
Lane 5: MCF7 cell lysate
Lane 6: NIH/3T3 cell lysate
Lane 7: PC-12 cell lysate
Lane 8: Mouse brain tissue lysate
Lane 9: Rat brain tissue lysate
Lane 10: 293T treated with λpp for 1 hour cell lysate
Lane 11: SH-SY5Y treated with λpp for 1 hour cell lysate

Lysates/proteins at 15 µg/Lane.

Predicted band size: 47/51 kDa
Observed band size: 47/51 kDa

Exposure time: 5 minutes;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1607-54) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:50,000 dilution was used for 1 hour at room temperature.
ET1607-54_2.jpg Fig2: ICC staining of Phospho-GSK3 (alpha + beta) (Y216 + Y279) in A431 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 10% negative goat serum for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1607-54, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 conjugate-Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
ET1607-54_3.jpg Fig3: ICC staining of Phospho-GSK3 (alpha + beta) (Y216 + Y279) in Hela cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 10% negative goat serum for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1607-54, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 conjugate-Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
ET1607-54_4.jpg Fig4: ICC staining of Phospho-GSK3 (alpha + beta) (Y216 + Y279) in MCF-7 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 10% negative goat serum for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1607-54, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 conjugate-Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
ET1607-54_5.jpg Fig5: Western blot analysis of Phospho-GSK3(alpha+beta)(Y216+Y279) on mouse cerebellum tissue lysates.

Lane 1: mouse cerebellum tissue, whole cell lysate, 20ug/lane
Lane 2: mouse cerebellum tissue treated with 2.8ug/ul lambda-PP for 30 minutes, whole cell lysates, 20ug/lane

All lanes :
Anti-Phospho-GSK3(alpha+beta)(Y216+Y279) antibody (ET1607-54) at 1:500 dilution. Anti-GAPDH antibody (ET1601-4) at 1:10,000 dilution. Goat Anti-Rabbit IgG H&L (HRP) (HA1001) at 1/200,000 dilution.

Predicted band size: 47/51 kDa
Observed band size: 47/51 kDa

Blocking and diluting buffer: 5% BSA.

Exposure time: 2 minutes
ET1607-54_6.jpg Fig6: Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-Phospho-GSK3 (alpha + beta) (Y216 + Y279) antibody (ET1607-54) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1607-54) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1607-54_7.jpg Fig7: Immunohistochemical analysis of paraffin-embedded rat hippocampus tissue with Rabbit anti-Phospho-GSK3 (alpha + beta) (Y216 + Y279) antibody (ET1607-54) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1607-54) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1607-54_8.jpg Fig8: Immunohistochemical analysis of paraffin-embedded mouse brain tissue with Rabbit anti-Phospho-GSK3 (alpha + beta) (Y216 + Y279) antibody (ET1607-54) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1607-54) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1607-54_9.jpg Fig9: Immunohistochemical analysis of paraffin-embedded human thyroid carcinoma tissue with Rabbit anti-Phospho-GSK3 (alpha + beta) (Y216 + Y279) antibody (ET1607-54) at 1/800 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1607-54) at 1/800 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1607-54_10.jpg Fig10: Flow cytometric analysis of HeLa cells labeling Phospho-GSK3 (alpha + beta) (Y216 + Y279).

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1607-54, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
ET1607-54_11.jpg Fig11: Flow cytometric analysis of NIH/3T3 cells labeling Phospho-GSK3 (alpha + beta) (Y216 + Y279).

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1607-54, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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