Revision 1

#48585Store at -20C

1 Kit

(4 x 20 microliters)

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.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Brg1 (D1Q7F) Rabbit mAb 49360 20 µl 220 kDa Rabbit IgG
BRD9 (E9R2I) Rabbit mAb 58906 20 µl 80 kDa Rabbit IgG
GLTSCR1 (E6I3A) Rabbit mAb 45441 20 µl 205 kDa Rabbit IgG
GLTSCR1L (E9K6F) Rabbit mAb 92805 20 µl 140 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

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Description

The Non-Canonical BAF Complex Antibody Sampler Kit provides an economical means of detecting non-canonical BAF complex members using antibodies. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

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 antibodies.

Background

ATP-dependent chromatin remodeling complexes play an essential role in the regulation of various nuclear processes, such as gene expression, DNA replication, and repair (1,2). The SWI/SNF chromatin remodeling complex consists of more than 10 subunits with a single molecule of the ATPase catalytic subunit BRM or BRG1, but not both. The activities of these two subunits drive the disruption of histone-DNA contacts that lead to changes in accessibility of crucial regulatory elements within chromatin (2-5). The BRM/BRG1 containing SWI/SNF complexes are recruited to target promoters by transcription factors, such as nuclear receptors, p53, RB, and BRCA1, to regulate gene activation, cell growth, the cell cycle, and differentiation processes (1,6-9).
GLTSCR1 and its paralog GLTSCR1L, along with BRD9, have been identified as unique subunits of the non-canonical BAF (ncBAF) or GBAF complex. This complex contains either GLTSCR1 or GLTSCR1L instead of an ARID subunit, while also lacking the BAF45, BAF47, and BAF57 subunits. GBAF maps to regions distinct from the PBAF and canonical BAF complexes and has also been shown to be a synthetic lethal target for BAF driven cancers such as synovial sarcomas and rhabdoid tumors (10-12).

  1. Ho, L. and Crabtree, G.R. (2010) Nature 463, 474-84.
  2. Becker, P.B. and Hörz, W. (2002) Annu Rev Biochem 71, 247-73.
  3. Eberharter, A. and Becker, P.B. (2004) J Cell Sci 117, 3707-11.
  4. Bowman, G.D. (2010) Curr Opin Struct Biol 20, 73-81.
  5. Gangaraju, V.K. and Bartholomew, B. (2007) Mutat Res 618, 3-17.
  6. Lessard, J.A. and Crabtree, G.R. (2010) Annu Rev Cell Dev Biol 26, 503-32.
  7. Morettini, S. et al. (2008) Front Biosci 13, 5522-32.
  8. Wolf, I.M. et al. (2008) J Cell Biochem 104, 1580-6.
  9. Simone, C. (2006) J Cell Physiol 207, 309-14.
  10. Alpsoy, A. and Dykhuizen, E.C. (2018) J Biol Chem 293, 3892-3903.
  11. Mashtalir, N. et al. (2018) Cell 175, 1272-1288.e20.
  12. Michel, B.C. et al. (2018) Nat Cell Biol 20, 1410-1420.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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    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.

    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.
    Revision 1
    #48585

    Non-Canonical BAF Complex Antibody Sampler Kit

    Non-Canonical BAF Complex Antibody Sampler Kit: Image 1 Expand Image
    Simple Western™ analysis of lysates (1.0 mg/mL) from HDLM-2 cells using BRD9 (E9R2I) Rabbit mAb #58906. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 2 Expand Image
    Immunoprecipitation of GLTSCR1L protein from Jurkat cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is GLTSCR1L (E9K6F) Rabbit mAb. Western blot analysis was performed using GLTSCR1L (E9K6F) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from various cell lines using GLTSCR1L (E9K6F) Rabbit mAb.
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from various cell lines using GLTSCR1 (E6I3A) Rabbit mAb (upper) or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower). GLTSCR1/GLTSCR1L double knockout (KO) cell lysates were isolated and provided by the Kadoch laboratory.
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 5 Expand Image
    CUT&RUN was performed with NCCIT cells and GLTSCR1 (E6I3A) 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 figure shows binding across TCF21 gene.
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from various cell lines using Brg1 (D1Q7F) Rabbit mAb.
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 7 Expand Image
    CUT&RUN was performed with NCCIT cells and Brg1 (D1Q7F) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Library was prepared using DNA Library Prep Kit for Illumina®(ChIP-seq, CUT&RUN) #56795. The figure shows binding across TNN gene.
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from various cell lines using BRD9 (E9R2I) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, OVCAR-4 cells are low for BRD9 expression and no BRD9 is detected in the 293T knockout (KO) cells. BRD9 KO cell lysates were isolated and provided by the Kadoch laboratory.
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 9 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from Jurkat cells, either wild-type (WT) or GLTSCR1/GLTSCR1L double knockout (KO), using GLTSCR1L (E9K6F) Rabbit mAb (upper) or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower). GLTSCR1/GLTSCR1L double KO cell lysates were isolated and provided by the Kadoch laboratory.
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 11 Expand Image
    Immunoprecipitation of GLTSCR1 protein from Jurkat cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is GLTSCR1 (E6I3A) Rabbit mAb. Western blot analysis was performed using GLTSCR1 (E6I3A) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 12 Expand Image
    CUT&RUN was performed with NCCIT cells and GLTSCR1 (E6I3A) 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 TCF21 gene (upper) and GATA6 gene (lower).
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 13 Expand Image
    Western blot analysis of extracts from various cell lines using Brg1 (D1Q7F) Rabbit mAb (upper) or BRM (D9E8B) XP® Rabbit mAb (lower).
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 14 Expand Image
    CUT&RUN was performed with NCCIT cells and Brg1 (D1Q7F) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Library was prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 1 (upper), including TNN gene (lower).
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 15 Expand Image
    Immunoprecipitation of BRD9 protein from HDLM-2 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is BRD9 (E9R2I) Rabbit mAb. Western blot analysis was performed using BRD9 (E9R2I) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 16 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from untreated NCCIT cells and either GLTSCR1 (E6I3A) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005.  The enriched DNA was quantified by real-time PCR using SimpleChIP® Human Oct-4 Promoter Primers #4641, SimpleChIP® Human Sox2 Promoter Primers #4649, 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.
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 17 Expand Image
    CUT&RUN was performed with NCCIT cells and either GLTSCR1 (E6I3A) 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 FOXP2 promoter primers, human YARS2 promoter primers, and human PXDNL 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.
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 18 Expand Image
    Immunoprecipitation of Brg1 from NCCIT cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Brg1 (D1Q7F) Rabbit mAb. Western blot analysis was performed using Brg1 (D1Q7F) Rabbit mAb.
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 19 Expand Image
    CUT&RUN was performed with NCCIT cells and either Brg1 (D1Q7F) 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 HoxA1 Intron 1 Primers #7707, SimpleChIP® Human HoxA2 Promoter Primers #5517, and human ITM2A upstream 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.
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 20 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 d followed by treatment with β-estradiol (10 nM, 45 min) and Brg1 (D1Q7F) XP® Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using DNA Library Prep Kit for Illumina®(ChIP-seq, CUT&RUN) #56795. The figure shows binding across the ENSA gene.
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 21 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 d followed by treatment with β-estradiol (10 nM, 45 min) and Brg1 (D1Q7F) XP® Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using DNA Library Prep Kit for Illumina®(ChIP-seq, CUT&RUN) #56795. The figure shows binding across ESR1 (upper), a known target gene of Brg1 (see additional figure containing ChIP-qPCR data), and ENSA gene (lower).
    Non-Canonical BAF Complex Antibody Sampler Kit: Image 22 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 d followed by treatment with β-estradiol (10 nM, 45 min) and either Brg1 (D1Q7F) 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 ESR1 Promoter Primers #9673, SimpleChIP® Human pS2 Promoter Primers #9702, 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.
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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.