Revision 4

#64828Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IP, ChIP, C&R

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

13

Source/Isotype:

Rabbit IgG

UniProt ID:

#P63272

Entrez-Gene Id:

6827

Product Information

Product Usage Information

For optimal ChIP 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.
Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:50
Chromatin IP 1:100
CUT&RUN 1:50

Storage

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.

Specificity / Sensitivity

SPT4 (D3P2W) Rabbit mAb recognizes endogenous levels of total SPT4 protein.

Species Reactivity:

Human, Mouse, Rat, Monkey

Species predicted to react based on 100% sequence homology

Hamster, S. cerevisiae

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human SPT4 protein.

Background

DRB-sensitivity inducing factor (DSIF), a heterodimer composed of SPT4 and SPT5, is capable of both facilitating and inhibiting RNA polymerase II (RNAPII) activity (1-3). DSIF, together with NELF (Negative Elongation Factor), inhibits RNAPII elongation, resulting in promoter proximal pausing of RNAPII as it awaits additional signaling to resume transcription (4). The release of promoter proximal pausing is signaled through phosphorylation of the RNAPII C-terminal domain (CTD) and NELF by positive transcription elongation factor (P-TEFb) (5). P-TEFb also phosphorylates SPT5 at Thr4 within the evolutionarily conserved heptapeptide repeat motif. This phosphorylation event switches DSIF from a transcriptional repressor to an activator where it becomes a critical factor for transcriptional elongation (6,7).

  1. Wada, T. et al. (1998) Genes Dev 12, 343-56.
  2. Saunders, A. et al. (2006) Nat Rev Mol Cell Biol 7, 557-67.
  3. Gomes, N.P. et al. (2006) Genes Dev 20, 601-12.
  4. Yamaguchi, Y. et al. (1999) Cell 97, 41-51.
  5. Buratowski, S. (2009) Mol Cell 36, 541-6.
  6. Wada, T. et al. (1998) EMBO J 17, 7395-403.
  7. Yamada, T. et al. (2006) Mol Cell 21, 227-37.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation ChIP: Chromatin IP C&R: CUT&RUN

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SimpleChIP is a registered trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Revision 4
#64828

SPT4 (D3P2W) Rabbit mAb

Western Blotting Image 1: SPT4 (D3P2W) Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using SPT4 (D3P2W) Rabbit mAb.
Immunoprecipitation Image 1: SPT4 (D3P2W) Rabbit mAb Expand Image
Immunoprecipitation of SPT4 from HCT 116 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is SPT4 (D3P2W) Rabbit mAb. Western blot analysis was performed using SPT4 (D3P2W) Rabbit mAb.
Chromatin Immunoprecipitation Image 1: SPT4 (D3P2W) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from HCT 116 cells and either SPT4 (D3P2W) 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 c-Myc Intron 1 Primers #14905, 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 and RUN Image 1: SPT4 (D3P2W) Rabbit mAb Expand Image
CUT&RUN was performed with HCT116 cells and SPT4 (D3P2W) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figures show binding across CALM2 gene, a known target gene of SPT4 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 2: SPT4 (D3P2W) Rabbit mAb Expand Image
CUT&RUN was performed with HCT116 cells and SPT4 (D3P2W) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 2 (upper), including CALM2 (lower), a known target gene of SPT4 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 3: SPT4 (D3P2W) Rabbit mAb Expand Image
CUT&RUN was performed with HCT116 cells and either SPT4 (D3P2W) 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 CALM2 promoter primers and human GALM promoter primers. 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.