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78779
PBAF Complex Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

PBAF Complex Antibody Sampler Kit #78779

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PBAF Complex Antibody Sampler Kit: Image 1

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 SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across the ENSA gene.

PBAF Complex Antibody Sampler Kit: Image 2

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using BRD7 (D9K2T) Rabbit mAb.

PBAF Complex Antibody Sampler Kit: Image 3

Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 days, then treated with β-estradiol (100 nM) for 45 minutes and either PBRM1/BAF180 (E9X2Z) 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 Human ESR1 Intron 1 Primers, SimpleChIP® Human CCND1 Promoter Primers #12531, 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.

PBAF Complex Antibody Sampler Kit: Image 4

Immunoprecipitation of ARID2 from Jurkat cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is ARID2 (D8D8U) Rabbit mAb. Western blot analysis was performed using ARID2 (D8D8U) Rabbit mAb.

PBAF Complex Antibody Sampler Kit: Image 5

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 SMARCC1/BAF155 (D7F8S) Rabbit mAb, SMARCB1/BAF47 (D8M1X) Rabbit mAb #91735, or SS18 (D6I4Z) Rabbit mAb #21792, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. SMARCC1/BAF155, SMARCB1/BAF47, and SS18 are all subunits of SWI/SNF complex. The figure shows binding across pS2/TFF1, a known target gene of SWI/SNF complex (see additional figure containing ChIP-qPCR data).

PBAF Complex Antibody Sampler Kit: Image 6

CUT&RUN was performed with NCCIT cells and SMARCC1/BAF155 (D7F8S) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across Sox2, a known target gene of SMARCC1 (see additional figure containing CUT&RUN-qPCR data).

PBAF Complex Antibody Sampler Kit: Image 7

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.

PBAF Complex Antibody Sampler Kit: Image 8

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 SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #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).

PBAF Complex Antibody Sampler Kit: Image 9

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using BRD7 (D9K2T) Rabbit mAb.

PBAF Complex Antibody Sampler Kit: Image 10

Immunoprecipitation of PBRM1/BAF180 protein from Jurkat cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) maAb IgG XP® Isotype Control #3900, and lane 3 is PBRM1/BAF180 (E9X2Z) Rabbit mAb. Western blot analysis was performed using PBRM1/BAF180 (E9X2Z) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.

PBAF Complex Antibody Sampler Kit: Image 11

Western blot analysis of extracts from various cell lines using ARID2 (D8D8U) Rabbit mAb (upper) and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).

PBAF Complex Antibody Sampler Kit: Image 12

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 SMARCC1/BAF155 (D7F8S) Rabbit mAb, SMARCB1/BAF47 (D8M1X) Rabbit mAb #91735, or SS18 (D6I4Z) Rabbit mAb #21792, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. SMARCC1/BAF155, SMARCB1/BAF47, and SS18 are all subunits of SWI/SNF complex. The figure shows binding across chromosome 21 (upper), including pS2/TFF1 (lower), a known target gene of SWI/SNF complex (see additional figure containing ChIP-qPCR data).

PBAF Complex Antibody Sampler Kit: Image 13

CUT&RUN was performed with NCCIT cells and SMARCC1/BAF155 (D7F8S) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figures show binding across chromosome 3 (upper), including Sox2 (lower), a known target gene of SMARCC1 (see additional figure containing CUT&RUN-qPCR data).

PBAF Complex Antibody Sampler Kit: Image 14

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.

PBAF Complex Antibody Sampler Kit: Image 15

Immunohistochemical analysis of paraffin-embedded MDA-MB-231 (left) and SW620 (right) cell pellets using BRD7 (D9K2T) Rabbit mAb.

PBAF Complex Antibody Sampler Kit: Image 16

Western blot analysis of extracts from various cell lines using PBRM1/BAF180 (E9X2Z) Rabbit mAb (upper) or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower). As expected, HCC1143 cells are negative for PBRM1/BAF180 expression.

PBAF Complex Antibody Sampler Kit: Image 17

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 SMARCC1/BAF155 (D7F8S) 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 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.

PBAF Complex Antibody Sampler Kit: Image 18

CUT&RUN was performed with NCCIT cells and either SMARCC1/BAF155 (D7F8S) 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 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.

PBAF Complex Antibody Sampler Kit: Image 19

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.

PBAF Complex Antibody Sampler Kit: Image 20

Western blot analysis of extracts from various cell lines using BRD7 (D9K2T) Rabbit mAb.

PBAF Complex Antibody Sampler Kit: Image 21

Western blot analysis of extracts from various cell lines using Brg1 (D1Q7F) Rabbit mAb.

PBAF Complex Antibody Sampler Kit: Image 22

Western blot analysis of extracts from various cell lines using Brg1 (D1Q7F) Rabbit mAb (upper) or BRM (D9E8B) XP® Rabbit mAb (lower).

PBAF Complex Antibody Sampler Kit: Image 23

Immunoprecipitation of SMARCC1/BAF155 from HeLa cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or SMARCC1/BAF155 (D7F8S) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using SMARCC1/BAF155 (D7F8S) Rabbit mAb.

PBAF Complex Antibody Sampler Kit: Image 24

Western blot analysis of extracts from various cell lines using SMARCC1/BAF155 (D7F8S) Rabbit mAb.

To Purchase # 78779T
Product # Size Price
78779T
1 Kit  (5 x 20 µl) $ 391

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Brg1 (D1Q7F) Rabbit mAb 49360 20 µl
  • WB
  • IP
  • ChIP
H M R Mk 220 Rabbit IgG
BRD7 (D9K2T) Rabbit mAb 15125 20 µl
  • WB
  • IHC
H 85 Rabbit IgG
PBRM1/BAF180 (E9X2Z) Rabbit mAb 89123 20 µl
  • WB
  • IP
  • ChIP
H M R Mk 205 Rabbit IgG
ARID2 (D8D8U) Rabbit mAb 82342 20 µl
  • WB
  • IP
H 220 Rabbit IgG
SMARCC1/BAF155 (D7F8S) Rabbit mAb 11956 20 µl
  • WB
  • IP
  • ChIP
  • C&R
H M R Mk 155 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The PBAF Complex Antibody Sampler Kit provides an economical means of detecting members of the PBAF chromatin remodeling complex. ARID2, BRD7, and PBRM1/BAF180 are unique members of the PBAF complex, while Brg1 and SMARCC1/BAF155 are shared with BAF and non-canonical BAF complexes. The kit includes enough antibody to perform two western blot experiments with each primary antibody.

Specificity / Sensitivity

Each antibody in the PBAF Complex Antibody Sampler Kit detects endogenous levels of its target protein. In addition, Brg1 (D1Q7F) Rabbit mAb does not cross-react with BRM protein.

Source / Purification

Monoclonal antibodies are produced by immunizing rabbits with synthetic peptides corresponding to residues surrounding Val891 of human ARID2, Leu1672 of human PBRM1/BAF180, and Gly975 of human SMARCC1/BAF155, recombinant protein specific to the carboxy terminus of human BRD7, and recombinant protein specific to the amino terminus of human Brg1.

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).

PBRM1/BAF180, ARID2, and BRD7 are unique members of the SWI/SNF complex PBAF, which binds to kinetochores in mitotic chromatin (10,11). PBAF is involved in nuclear receptor-mediated transcription and retinoic acid driven gene activation (12,13). PBRM1/BAF180 has been shown to be a potent tumor suppressor, as it is the second-most mutated gene in renal carcinomas (14). ARID2 is the targeting subunit of the PBAF complex and critical for complex stability (15). Bromodomain-containing BRD7 interacts with acetylated histones to regulate gene transcription (16,17). SMARCC1/BAF155 is a core subunit of all BAF complexes including PBAF, and is necessary for efficient nucleosome remodeling by BRG1 in vitro (18).

  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. Nie, Z. et al. (2000) Mol Cell Biol 20, 8879-88.
  11. Xue, Y. et al. (2000) Proc Natl Acad Sci U S A 97, 13015-20.
  12. Lemon, B. et al. Nature 414, 924-8.
  13. Wang, Z. et al. (2004) Genes Dev 18, 3106-16.
  14. Varela, I. et al. (2011) Nature 469, 539-42.
  15. Yan, Z. et al. (2005) Genes Dev 19, 1662-7.
  16. Peng, C. et al. (2006) J Cell Biochem 97, 882-92.
  17. Kaeser, M.D. et al. (2008) J Biol Chem 283, 32254-63.
  18. Phelan, M.L. et al. (1999) Mol Cell 3, 247-53.

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