Figure 1: Treatment of NIH/3T3 cells with Calyculin A causes accumulation of phospho-histone H3 (Ser10), detected by Sandwich ELISA kit #7155, but does not affect the level of total histone H3 protein, detected by western analysis. OD 450 readings are shown in the top figure, while the corresponding western blot using Phospho-Histone H3 (Ser10) Antibody #9701 or Histone H3 Antibody #9715, is shown in the bottom figure.
Figure 2: Linear relationship between protein concentration of lysates from untreated and Calyculin A-treated NIH/3T3 cells and kit assay optical density readings. NIH/3T3 cells (80% confluence) were serum-starved overnight, and serum was added back for 15 minutes followed by treatment with Calyculin A (0.1 μM for 15 minutes).
|Product Includes||Volume||Solution Color|
|Histone H3 Rabbit mAb Coated Microwells||96 tests|
|Phospho-Histone H3 (Ser10) Rabbit Detection mAb (Biotinylated)||1 ea||Green (Lyophilized)|
|HRP-Linked Streptavidin (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
CST's PathScan® Phospho-Histone H3 (Ser10) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Phospho-Histone H3 (Ser10) protein. A Histone H3 Antibody has been coated onto the microwells. After incubation with cell lysates, both nonphospho- and phospho-Histone H3 proteins are captured by the coated antibody. Following extensive washing, a Biotinylated Phospho-Histone H3 (Ser10) Antibody is added to detect the captured phospho-Histone H3 (Ser10) protein. HRP-linked Streptavidin is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of Phospho-Histone H3 (Ser10) protein.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
CST's PathScan® Phospho-Histone H3 (Ser10) Sandwich ELISA Kit detects endogenous levels of Phospho-Histone H3 (Ser10). Using this Sandwich ELISA Kit #7155, Phospho-Histone H3 (Ser10) is detected when treated with Calyculin A in NIH/3T3 cells. However, the levels of Histone H3 remains unchanged, as shown by western analysis using the Histone H3 Antibody #9715 (Figure 1). 293 cells treated with Calyculin A show similar results (data not shown). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Modulation of chromatin structure plays an important role in the regulation of transcription in eukaryotes. The nucleosome, made up of DNA wound around eight core histone proteins (two each of H2A, H2B, H3, and H4), is the primary building block of chromatin (1). The amino-terminal tails of core histones undergo various post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitination (2-5). These modifications occur in response to various stimuli and have a direct effect on the accessibility of chromatin to transcription factors and, therefore, gene expression (6). In most species, histone H2B is primarily acetylated at Lys5, 12, 15, and 20 (4,7). Histone H3 is primarily acetylated at Lys9, 14, 18, 23, 27, and 56. Acetylation of H3 at Lys9 appears to have a dominant role in histone deposition and chromatin assembly in some organisms (2,3). Phosphorylation at Ser10, Ser28, and Thr11 of histone H3 is tightly correlated with chromosome condensation during both mitosis and meiosis (8-10). Phosphorylation at Thr3 of histone H3 is highly conserved among many species and is catalyzed by the kinase haspin. Immunostaining with phospho-specific antibodies in mammalian cells reveals mitotic phosphorylation at Thr3 of H3 in prophase and its dephosphorylation during anaphase (11).
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