HA722443

Phospho-SMAD2 (S465+467) Recombinant Rabbit Monoclonal Antibody [PSH05-76]

cat.: HA722443

Product Type:	Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity:	Human, Mouse, Rat
Applications:	WB, IF-Cell, IHC-P
Clonality:	Monoclonal
Clone number:	PSH05-76

Form:	Liquid
Storage condition:	Shipped at 4℃. Store at +4℃ short term (1-2 weeks). It is recommended to aliquot into single-use upon delivery. Store at -20℃ long term.
Storage buffer:	PBS (pH7.4), 0.1% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration:	1ug/ul
Purification:	Protein A affinity purified.
Molecular weight:	Predicted band size: 52/48 kDa
Isotype:	IgG
Immunogen:	Synthetic phospho-peptide corresponding to residues surrounding Ser465 and 467 of Human SMAD2.
Positive control:	HeLa cell lysate, HeLa starved overnight then treated with 10ng/mL TGF-β1 for 30 minutes cell lysate, NIH/3T3 starved overnight then treated with 10ng/mL TGF-β1 for 30 minutes cell lysate, HeLa cells treated with 20ng/mL TGF-β1 for 15 minutes, HeLa, NIH/3T3, human stomach tissue, rat lung tissue.
Subcellular location:	Cytoplasm, Nucleus.
Recommended Dilutions: WB IF-Cell IHC-P	1:1,000 1:500 1:200
Uniprot #:	SwissProt: Q15796 Human \| Q62432 Mouse \| O70436 Rat
Alternative names:	Drosophila, homolog of, MADR2 hMAD-2 HsMAD2 JV18 JV18-1 JV181 MAD MAD homolog 2 MAD Related Protein 2 Mad-related protein 2 MADH2 MADR2 MGC22139 MGC34440 Mother against DPP homolog 2 Mothers against decapentaplegic homolog 2 Mothers against decapentaplegic, Drosophila, homolog of, 2 Mothers against DPP homolog 2 OTTHUMP00000163489 Sma and Mad related protein 2 Sma- and Mad-related protein 2 MAD SMAD 2 SMAD family member 2 SMAD, mothers against DPP homolog 2 SMAD2 SMAD2_HUMAN

Images

Fig1: Western blot analysis of Phospho-SMAD2 (S465+467) on different lysates with Rabbit anti-Phospho-SMAD2 (S465+467) antibody (HA722443) at 1/1,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HeLa starved overnight then treated with 10ng/mL TGF-β1 for 30 minutes cell lysate
Lane 3: NIH/3T3 cell lysate
Lane 4: NIH/3T3 starved overnight then treated with 10ng/mL TGF-β1 for 30 minutes cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 52/48 kDa
Observed band size: 60 kDa

Exposure time: 1 minute 20 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 (HA722443) at 1/1,000 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.

Fig2: Immunocytochemistry analysis of HeLa cells treated with or without 20ng/mL TGF-β1 for 15 minutes labeling Phospho-SMAD2 (S465+467) with Rabbit anti-Phospho-SMAD2 (S465+467) antibody (HA722443) 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-SMAD2 (S465+467) antibody (HA722443) 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.

	Fig3: Immunocytochemistry analysis of NIH/3T3 cells treated with or without 20ng/mL TGF-β1 for 15 minutes labeling Phospho-SMAD2 (S465+467) with Rabbit anti-Phospho-SMAD2 (S465+467) antibody (HA722443) 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-SMAD2 (S465+467) antibody (HA722443) 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: Immunohistochemical analysis of paraffin-embedded human stomach tissue with Rabbit anti-Phospho-SMAD2 (S465+467) antibody (HA722443) at 1/200 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 (HA722443) at 1/200 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.
	Fig5: Immunohistochemical analysis of paraffin-embedded rat lung tissue with Rabbit anti-Phospho-SMAD2 (S465+467) antibody (HA722443) at 1/200 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 (HA722443) at 1/200 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.

Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.