HA750128

Phospho-GSK3 beta (S9) Recombinant Rabbit Monoclonal Antibody [SY02-71]

cat.: HA750128

Product Type:	Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity:	Human, Mouse, Rat
Applications:	WB, IF-Cell, IF-Tissue, IHC-P, FC
Clonality:	Monoclonal
Clone number:	SY02-71

Form:	Liquid
Storage condition:	Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer:	1*PBS (pH7.4).
Concentration:	1ug/ul
Purification:	Protein A affinity purified.
Molecular weight:	Predicted band size: 47 kDa
Isotype:	IgG
Immunogen:	Synthetic phospho-peptide corresponding to residues surrounding Ser9 of Human GSK3 beta aa 1-50 / 420.
Positive control:	HeLa treated with 100nM Calyculin A for 30 minutes cell lysate, NIH/3T3 cell lysate, NIH/3T3 treated with 100ng/mL PDGF for 5 minutes cell lysate, C6 cell lysate, C6 treated with 100ng/mL PDGF for 5 minutes cell lysate, C6 treated with 150nM insulin for 15 minutes cell lysate, HeLa cells treated with 100nM Calyculin A for 30 minutes, NIH/3T3 cells treated with 100ng/mL PDGF for 5 minutes, C6 cells treated with 100ng/mL PDGF for 5 minutes, human colon carcinoma tissue, human breast tissue, human breast carcinoma tissue, human kidney tissue, human pancreas tissue.
Subcellular location:	Cytoplasm, Nucleus, Cell membrane.
Recommended Dilutions: WB IF-Cell IF-Tissue IHC-P FC	1:5,000-1:10,000 1:100-1:500 1:50-1:200 1:50-1:200 1:1,000
Uniprot #:	SwissProt: P49841 Human \| Q9WV60 Mouse \| P18266 Rat
Alternative names:	Glycogen Synthase Kinase 3 Beta Glycogen synthase kinase-3 beta GSK 3 beta GSK-3 beta GSK3B GSK3B_HUMAN GSK3beta isoform Serine/threonine-protein kinase GSK3B

Images

Fig1: Western blot analysis of Phospho-GSK3 beta (S9) on different lysates with Rabbit anti-Phospho-GSK3 beta (S9) antibody (HA750128) at 1/5,000 dilution and competitor's antibody at 1/1,000 dilution.

Lane 1: HeLa cell lysate (15 µg/Lane)
Lane 2: HeLa treated with 100nM Calyculin A for 30 minutes cell lysate (15 µg/Lane)

Predicted band size: 47 kDa
Observed band size: 47 kDa

Exposure time: Lane 1-2 (left): 46 seconds; ECL: K1801;
Exposure time: Lane 1-2 (right): 45 seconds; ECL: K1802;
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 (HA750128) at 1/5,000 dilution and competitor's antibody at 1/1,000 dilution were 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.

Fig2: Western blot analysis of Phospho-GSK3 beta (S9) on different lysates with Rabbit anti-Phospho-GSK3 beta (S9) antibody (HA750128) at 1/5,000 dilution.

Lane 1: NIH/3T3 cell lysate (20 µg/Lane)
Lane 2: NIH/3T3 treated with 100ng/mL PDGF for 5 minutes cell lysate (20 µg/Lane)
Lane 3: C6 cell lysate (20 µg/Lane)
Lane 4: C6 treated with 100ng/mL PDGF for 5 minutes cell lysate (20 µg/Lane)
Lane 5: C6 cell lysate (20 µg/Lane)
Lane 6: C6 treated with 150nM insulin for 15 minutes cell lysate (20 µg/Lane)

Predicted band size: 47 kDa
Observed band size: 47 kDa
Exposure time: 6 seconds; ECL: K1801;
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 (HA750128) 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.

Fig3: Immunocytochemistry analysis of HeLa cells treated with 100nM Calyculin A for 30 minutes labeling Phospho-GSK3 beta (S9) with Rabbit anti-Phospho-GSK3 beta (S9) antibody (HA750128) at 1/500 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-GSK3 beta (S9) antibody (HA750128) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Fig4: Immunocytochemistry analysis of NIH/3T3 cells treated with 100ng/mL PDGF for 5 minutes labeling Phospho-GSK3 beta (S9) with Rabbit anti-Phospho-GSK3 beta (S9) antibody (HA750128) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-GSK3 beta (S9) antibody (HA750128) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

	Fig5: Immunocytochemistry analysis of C6 cells treated with 100ng/mL PDGF for 5 minutes labeling Phospho-GSK3 beta (S9) with Rabbit anti-Phospho-GSK3 beta (S9) antibody (HA750128) at 1/100 dilution. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-GSK3 beta (S9) antibody (HA750128) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
	Fig6: Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue with Rabbit anti-Phospho-GSK3 beta (S9) antibody (HA750128) at 1/50 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 (HA750128) at 1/50 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.
	Fig7: Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-Phospho-GSK3 beta (S9) antibody (HA750128) at 1/50 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 (HA750128) at 1/50 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.

Fig8: Flow cytometric analysis of NIH/3T3 cells treated with 100ng/mL PDGF for 5 minutes labeling Phospho-GSK3 beta (S9).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA750128, 1/1,000) (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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