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Acetyl-Histone H3 (Lys27) (D5E4) XP ® Rabbit mAb
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Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding acetylated Lys27 of human histone H3 protein.
Product Usage Information
For optimal ChIP and ChIP-seq results, use 5 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.
The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
The CUT&Tag dilution was determined using CUT&Tag Assay Kit #77552.
| Application | Dilution |
|---|---|
| Western Blotting | 1:1000 |
| Simple Western™ | 1:250 |
| Immunofluorescence (Immunocytochemistry) | 1:50 - 1:200 |
| Flow Cytometry (Fixed/Permeabilized) | 1:50 - 1:200 |
| Chromatin IP | 1:100 |
| Chromatin IP-seq | 1:100 |
| CUT&RUN | 1:100 |
| CUT&Tag | 1:50 |
Specificity/Sensitivity
Species Reactivity:
Human, Mouse, Rat, Monkey
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For a carrier free (BSA and azide free) version of this product see product #87261.
参考图片
Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb specificity was determined by peptide ELISA. The graph depicts the binding of the antibody to precoated acetyl-histone H3 (Lys27) peptide in the presence of increasing concentrations of various competitor peptides. As shown, only the acetyl-histone H3 (Lys27) peptide competed away binding of the antibody.
Western blot analysis of extracts from HeLa and C2C12 cells, untreated (-) or treated (+) with Trichostatin A (TSA) #9950 (1 μM, 18 hr), using Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb (upper) or Histone H3 (D1H2) XP® Rabbit mAb #4499 (lower).
Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with Trichostatin A (TSA) #9950 (1 uM, 4 hr; right), using Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).
Flow cytometric analysis of HeLa cells, untreated (blue) or treated with Trichostatin A (TSA) #9950 (1 μM, 18 hr; green) using Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across GAPDH, a known target gene of H3K27Ac (see additional figure containing ChIP-qPCR data).
Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 12 (upper), including GAPDH (lower), a known target gene of H3K27Ac (see additional figure containing ChIP-qPCR data).
Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and either Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human GAPDH Exon 1 Primers #5516, SimpleChIP® Human RPL30 Exon 3 Primers #7014, SimpleChIP® Human AFM Intron 1 Primers #5098, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
CUT&RUN was performed with HeLa cells and Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figures show binding across GAPDH, a known target gene of H3K27Ac (see additional figure containing CUT&RUN-qPCR data).
CUT&RUN was performed with HeLa cells and Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 12 (upper), including GAPDH (lower), a known target gene of H3K27Ac (see additional figure containing CUT&RUN-qPCR data).
CUT&RUN was performed with HeLa cells and either Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human RPL30 Exon 3 Primers #7014, SimpleChIP® Human GAPDH Exon 1 Primers #5516, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
CUT&Tag was performed with HeLa cells and Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figure shows binding across GAPDH, a known target gene of H3K27ac (see our ChIP-qPCR figure).
CUT&Tag was performed with HeLa cells and Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figures show binding across chromosome 12 (upper), including GAPDH (lower), a known target gene of H3K27ac (see our ChIP-qPCR figure).