WB result of Phospho-Histone H3 (Ser28) Recombinant Rabbit mAb
Primary antibody: Phospho-Histone H3 (Ser28) Recombinant Rabbit mAb at 1/1000 dilution
Lane 1: untreated HeLa whole cell lysate 20 µg
Lane 2: HeLa treated with 100 ng/ml Colcemid for 16 hours whole cell lysate 20 µg
Secondary antibody: Goat Anti-rabbit IgG, (H+L), HRP conjugated at 1/10000 dilution
Predicted MW: 15 kDa
Observed MW: 14 kDa
Phospho-Histone H3 (Ser28) Recombinant Rabbit mAb (S-1025-45)
Phospho-Histone H3 (Ser28) Recombinant Rabbit mAb (S-1025-45)
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Product Details
Product Details
Product Specification
| Host | Rabbit |
| Antigen | Phospho-Histone H3 (Ser28) |
| Synonyms | H3S28P |
| Immunogen | Synthetic Peptide |
| Location | Nucleus |
| Accession | P68431 |
| Clone Number | S-1025-45 |
| Antibody Type | Recombinant mAb |
| Isotype | IgG |
| Application | WB, IHC-P, IP, ChIP |
| Reactivity | Hu, Ms, Rt |
| Purification | Protein A |
| Concentration | 0.5 mg/ml |
| Conjugation | Unconjugated |
| Physical Appearance | Liquid |
| Storage Buffer | PBS, 40% Glycerol, 0.05% BSA, 0.03% Proclin 300 |
| Stability & Storage | 12 months from date of receipt / reconstitution, -20 °C as supplied |
Dilution
| application | dilution | species |
| Dot Blot | 1:1000 | |
| WB | 1:1000 | |
| IP | 1:50 | |
| IHC-P | 1:250 | |
| ChIP | 1:20-1:50 |
Background
H3S28P is an epigenetic modification to the DNA packaging protein histone H3. It is a mark that indicates the phosphorylation the 28th serine residue of the histone H3 protein. Early mitosis phosphorylation patterns for H3S10 and H3S28 are quite similar, starting at the initiation of chromosomal condensation during prophase. At gene promoters, H3S28 phosphorylation is expected to remove Polycomb restrictive complexes from chromatin, causing demethylation and acetylation of the nearby K27 residue, so activating transcription. This transition is mediated by the phosphorylation of H3S28 on a site next to H3K27. It was discovered that phosphorylating H3S28 in the promoters of genes like c-fos and -globin controlled their activation.
Picture
Picture
Western Blot
IP
Phospho-Histone H3 (Ser28) Rabbit mAb at 1/50 dilution (1 µg) immunoprecipitating Phospho-Histone H3 (Ser28) in 0.4 mg HeLa treated with 100 ng/ml Colcemid for 16 hours whole cell lysate.
Western blot was performed on the immunoprecipitate using Phospho-Histone H3 (Ser28) Rabbit mAb at 1/1000 dilution.
Secondary antibody (HRP) for IP was used at 1/1000 dilution.
Lane 1: HeLa treated with 100 ng/ml Colcemid for 16 hours whole cell lysate 20 µg (Input)
Lane 2: Phospho-Histone H3 (Ser28) Rabbit mAb IP in HeLa treated with 100 ng/ml Colcemid for 16 hours whole cell lysate
Lane 3: Rabbit monoclonal IgG IP in HeLa treated with 100 ng/ml Colcemid for 16 hours whole cell lysate
Predicted MW: 15 kDa
Observed MW: 14 kDa
Dot Blot
Dot blot result of Phospho-Histone H3 (Ser28) Recombinant Rabbit mAb
Lane 1: Phospho-Histone H3 (Ser28) peptide
Lane 2: Histone H3 unmodified peptide
Primary antibody: Phospho-Histone H3 (Ser28) Recombinant Rabbit mAb at 1/1000 dilution
Secondary antibody: Goat Anti-rabbit IgG, (H+L), HRP conjugated at 1/10000 dilution
Immunohistochemistry
IHC shows positive staining in paraffin-embedded human colon. Anti- Phospho-Histone H3 (Ser28) antibody was used at 1/250 dilution, followed by a HRP Polymer for Mouse & Rabbit IgG (ready to use). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris/EDTA buffer pH9.0 was performed before commencing with IHC staining protocol.
IHC shows positive staining in paraffin-embedded human lung. Anti- Phospho-Histone H3 (Ser28) antibody was used at 1/250 dilution, followed by a HRP Polymer for Mouse & Rabbit IgG (ready to use). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris/EDTA buffer pH9.0 was performed before commencing with IHC staining protocol.
IHC shows positive staining in paraffin-embedded human breast cancer. Anti- Phospho-Histone H3 (Ser28) antibody was used at 1/250 dilution, followed by a HRP Polymer for Mouse & Rabbit IgG (ready to use). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris/EDTA buffer pH9.0 was performed before commencing with IHC staining protocol.
IHC shows positive staining in paraffin-embedded human colon cancer. Anti- Phospho-Histone H3 (Ser28) antibody was used at 1/250 dilution, followed by a HRP Polymer for Mouse & Rabbit IgG (ready to use). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris/EDTA buffer pH9.0 was performed before commencing with IHC staining protocol.
IHC shows positive staining in paraffin-embedded mouse colon. Anti- Phospho-Histone H3 (Ser28) antibody was used at 1/250 dilution, followed by a HRP Polymer for Mouse & Rabbit IgG (ready to use). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris/EDTA buffer pH9.0 was performed before commencing with IHC staining protocol.
IHC shows positive staining in paraffin-embedded rat colon. Anti- Phospho-Histone H3 (Ser28) antibody was used at 1/250 dilution, followed by a HRP Polymer for Mouse & Rabbit IgG (ready to use). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris/EDTA buffer pH9.0 was performed before commencing with IHC staining protocol.
ChIP
Chromatin immunoprecipitation (ChIP) was performed on HeLa + Nocodazole (100ng/ml,18h) cells cross - linked with 1% formaldehyde for 10 min, then chromatin was fragmented by sonication.
Parallel reactions used Phospho-Histone H3 (Ser28) Recombinant Rabbit mAb (S-1025-45) and Rabbit mAb IgG Isotype Control (SDT-R173) at 1:50 for immunoprecipitation.
Post - immunoprecipitation, both samples were washed, eluted, and cross - links reversed. Purified DNA was analyzed by qPCR.
qPCR (%input: immunoprecipitated DNA/input DNA)
showed the enrichment of RPL30, GAPDH, AFM, MYOD1,
SAT-α and SAT-2 in Phospho-Histone H3 (Ser28)
Recombinant Rabbit mAb (S-1025-45)-immunoprecipitated
sample.
