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ATF-4 (D4B8) Rabbit mAb
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Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human ATF-4 protein.
Product Usage Information
For optimal ChIP and ChIP-seq results, use 2.5 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 the CUT&RUN Assay Kit #86652.
The CUT&Tag dilution was determined using CUT&Tag Assay Kit #77552.
| Application | Dilution |
|---|---|
| Western Blotting | 1:1000 |
| Immunoprecipitation | 1:50 |
| Immunofluorescence (Immunocytochemistry) | 1:100 - 1:400 |
| Chromatin IP | 1:200 |
| Chromatin IP-seq | 1:200 |
| CUT&RUN | 1:200 |
| CUT&Tag | 1:200 |
Specificity/Sensitivity
Species Reactivity:
Human, Mouse, Rat
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.
参考图片
CUT&Tag was performed with Hep G2 cells treated with Thapsigargin #12758 (300nM) for 4h and ATF-4 (D4B8) 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 Ddit3/CHOP (lower), a known target gene of ATF-4 (see our ChIP-qPCR figure).
Western blot analysis of extracts from 293 and HeLa cells, untreated (-) or tunicamycin-treated (2 μg/ml, 8 hr; +), using ATF-4 (D4B8) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Immunoprecipitation of ATF-4 from extracts of 293 cells, treated with tunicamycin (2 μg/ml, 8 hr), using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or ATF-4 (D4B8) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using ATF-4 (D4B8) Rabbit mAb. Mouse Anti-rabbit IgG (Light-Chain Specific) (L57A3) mAb #3677 and Anti-mouse IgG, HRP-linked Antibody #7076 were used as secondary antibodies.
Confocal immunofluorescent analysis of HeLa cells, untreated (left) or tunicamycin-treated (2 μg/ml, 8 hr; right), using ATF-4 (D4B8) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).
Chromatin immunoprecipitations were performed with cross-linked chromatin from mouse embryonic fibroblasts treated with tunicamycin (2ug/ml) overnight and ATF-4 (D4B8) Rabbit mAb, using SimpleChIP® 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 Ddit3/CHOP, a known target gene of ATF4 (see additional figure containing ChIP-qPCR data).
Chromatin immunoprecipitations were performed with cross-linked chromatin from mouse embryonic fibroblasts treated with tunicamycin (2ug/ml) overnight and ATF-4 (D4B8) Rabbit mAb, using SimpleChIP® 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 10 (upper), including Ddit3/CHOP (lower), a known target gene of ATF4 (see additional figure containing ChIP-qPCR data).
Chromatin immunoprecipitations were performed with cross-linked chromatin from mouse embryonic fibroblasts treated with tunicamycin (2ug/ml) overnight, and ATF-4 (D4B8) 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® Mouse ATF-3 Intron 1 Primers #13059, mouse CHOP promoter primers, and SimpleChIP® Mouse RPL30 Intron 2 Primers #7015. 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 Hep G2 cells treated with Thapsigargin #12758 (300nM) for 4h and ATF-4 (D4B8) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Library was prepared using DNA Library Prep Kit for Illumina® #56795. The figure shows binding across ASNS, a known target gene of ATF-4 (see additional figure containing CUT&RUN-qPCR data).
CUT&RUN was performed with Hep G2 cells treated with Thapsigargin #12758 (300nM) for 4h and ATF-4 (D4B8) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Library was prepared using DNA Library Prep Kit for Illumina® #56795. The figures show binding across chromosome 7 (upper), including ASNS (lower), a known target gene of ATF-4 (see additional figure containing CUT&RUN-qPCR data).
CUT&RUN was performed with Hep G2 cells treated with Thapsigargin #12758 (300nM) for 4h and either ATF-4 (D4B8) 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 human TRB3 exon 1 primer, human ASNS exon 1 primer 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 Hep G2 cells treated with Thapsigargin #12758 (300nM) for 4h and ATF-4 (D4B8) 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 Ddit3/CHOP gene, a known target gene of ATF4 (see our ChIP-qPCR figure).