ET1601-30

Phospho-Histone H3 (S10) Recombinant Rabbit Monoclonal Antibody [SA31-01]

cat.: ET1601-30

Product Type:	Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity:	Human, Mouse, Rat
Applications:	WB, IF-Cell, IF-Tissue, IHC-P, IP, FC
Clonality:	Monoclonal
Clone number:	SA31-01

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: 15 kDa
Isotype:	IgG
Immunogen:	Synthetic phospho-peptide corresponding to residues surrounding Ser10 of human Histone H3.
Positive control:	HeLa treated with 50nM Calyculin A for 30 minutes whole cell lysate, NIH/3T3 treated with 100nM Calyculin A for 30 minutes whole cell lysate, C6 treated with 100nM Calyculin A for 30 minutes whole cell lysate, HeLa, AGS, HepG2, mouse testis tissue.
Subcellular location:	Nucleus, Chromosome
Recommended Dilutions: WB IF-Cell IF-Tissue IHC-P IP FC	1:2,000-1:10,000 1:5,000 1:50 1:50 Use at an assay dependent concentration. 1:1,000
Uniprot #:	SwissProt: P68431 Human \| P84243 Human \| Q16695 Human \| Q6NXT2 Human \| Q71DI3 Human \| P84244 Mouse \| P84245 Rat
Alternative names:	H3 3 like sequence MH921 H3 3A H3 a H3 b H3 c H3 d H3 f H3 h H3 histone family member E pseudogene H3 i H3 j H3 k H3 l H33_HUMAN H3F3 H3f3b Histone H3 3 pseudogene Histone H3.3

Images

Fig1: Western blot analysis of Phospho-Histone H3 (S10) on different lysates with Rabbit anti-Phospho-Histone H3 (S10) antibody (ET1601-30) at 1/5,000 dilution.

Lane 1: HeLa whole cell lysate (20 µg/Lane)
Lane 2: HeLa treated with 50nM Calyculin A for 30 minutes whole cell lysate (20 µg/Lane)
Lane 3: NIH/3T3 whole cell lysate (20 µg/Lane)
Lane 4: NIH/3T3 treated with 100nM Calyculin A for 30 minutes whole cell lysate (20 µg/Lane)
Lane 5: C6 whole cell lysate (20 µg/Lane)
Lane 6: C6 treated with 100nM Calyculin A for 30 minutes whole cell lysate (20 µg/Lane)

Predicted band size: 15 kDa
Observed band size: 15 kDa

Exposure time: 10 seconds;

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 (ET1601-30) 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.

Fig2: Immunocytochemistry analysis of HeLa cells labeling Phospho-Histone H3 (S10) with Rabbit anti-Phospho-Histone H3 (S10) antibody (ET1601-30) at 1/5,000 dilution and competitor's antibody at 1/3,000 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-Histone H3 (S10) antibody (ET1601-30) at 1/5,000 dilution and competitor's antibody at 1/3,000 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: Western blot analysis of Phospho-Histone H3 (S10) on different lysates with Rabbit anti-Phospho-Histone H3 (S10) antibody (ET1601-30) at 1/2,000 dilution and competitor's antibody at 1/1,000 dilution.

Lane 1: HeLa whole cell lysate
Lane 2: HeLa treated with 50nM Calyculin A for 30 minutes whole cell lysate
Lane 3: C6 whole cell lysate
Lane 4: C6 treated with 100ng/mL Calyculin A for 1 hour whole cell lysate
Lane 5: NIH/3T3 whole cell lysate
Lane 6: NIH/3T3 starved for 4 hours, then treated with 100nM Calyculin A for 30 minutes whole cell lysate

Lysates/proteins at 15 µg/Lane.

Predicted band size: 15 kDa
Observed band size: 15 kDa

Exposure time: 15 seconds;

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 (ET1601-30) at 1/2,000 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.

Fig4: ICC staining of Phospho-Histone H3 (S10) in AGS cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1601-30, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

	Fig5: ICC staining of Phospho-Histone H3 (S10) in HepG2 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1601-30, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
	Fig6: Immunohistochemical analysis of paraffin-embedded mouse testis tissue using anti-Phospho-Histone H3 (S10) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1601-30, 1/50) for 30 minutes 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: Flow cytometric analysis of HeLa untreated cells and HeLa treated with 100nM Calyculin A for 30 minutes cells labeling Phospho-Histone H3 (S10). Cells were fixed and permeabilized. Then stained with the primary antibody (ET1601-30, 1μg/mL) (HeLa untreated，blue; HeLa treated, red) compared with Rabbit IgG Isotype Control (grey). 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).

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