Figure 1. Treatment of NIH/3T3 cells with trichostatin A (TSA) increases the acetylation of Histone H4 at Lys12 detected by PathScan® Acetyl-Histone H4 (Lys12) Sandwich ELISA Kit #7228. TSA treatment does not affect the level of histone H4 that is detected by Western analysis. NIH/3T3 cells (70-80% confluent) were treated for 16-18 hours with 0.4 μM TSA at 37ºC. Absorbance readings at 450 nm are shown in the top figure while the corresponding Western blots using Histone H4 Antibody #2592 (left panel) or Acetyl-Histone H4 (Lys12) Antibody #2591 (right panel) are shown in the bottom figure.
Figure 2: The relationship between protein concentration of lysates from untreated and TSA treated HeLa cells and kit assay optical density readings. HeLa cells were treated with TSA (4μM overnight). An acid extraction was performed for cell lysis in the presence of 5mM sodium butyrate.
|Product Includes||Volume||Solution Color|
|Histone H4 Mouse mAb Coated Microwells||96 tests|
|Acetyl-Histone H4 (Lys12) Rabbit Detection mAb||1 ea||Green (Lyophilized)|
|Anti-rabbit IgG, HRP-linked Antibody (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|
The PathScan® Acetyl-Histone H4 (Lys12) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of histone H4 when acetylated at lysine 12. A histone H4 mouse antibody has been coated onto the microwells. After incubation with cell lysates, histone H4 protein (acetylated and non-acetylated) is captured by the coated antibody. Following extensive washing, Acetyl-histone H4 (Lys12) rabbit antibody is added to detect acetylated Lys12 on the histone H4 protein. Anti-rabbit IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of histone H4 acetylated at Lys12.
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® Acetyl-Histone H4 (Lys12) Sandwich ELISA Kit #7228 detects endogenous levels of histone H4 when acetylated at Lys12. As shown in Figure 1 using the Acetyl-Histone H4 (Lys12) Sandwich ELISA Kit #7228, a high level of acetylation at Lys12 on histone H4 is detected in NIH/3T3 cells when treated with TSA. The level of total histone H4 (acetylated and non-acetylated) remains unchanged as shown by Western analysis (Figure 1). Similar results are obtained when COS and Jurkat cells are treated with TSA (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.Species Reactivity:
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).
Explore pathways + proteins related to this product.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a trademark of Cell Signaling Technology, Inc.