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12854
BAF Complex Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

BAF Complex Antibody Sampler Kit #12854

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BAF Complex Antibody Sampler Kit: Image 1

Confocal immunofluorescent analysis of 293 cells using Brg1 (A52) Antibody (green). Actin filaments have been labeled using DY-554 phalloidin (red).

BAF Complex Antibody Sampler Kit: Image 2

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 BRM (D9E8B) 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.

BAF Complex Antibody Sampler Kit: Image 3

colon:

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using BRM (D9E8B) XP(R) Rabbit mAb.

BAF Complex Antibody Sampler Kit: Image 4

Western blot analysis of extracts from control HeLa cells (Lane 1) or HeLa cells with a targeted mutation in the gene encoding BRM (Lane 2) using BRM (D9E8B) XP® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The change in BRM molecular weight in the mutated HeLa cells confirms the specificity of the antibody for BRM.

BAF 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 #11956, 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). For additional ChIP-seq tracks, please download the product data sheet.

BAF Complex Antibody Sampler Kit: Image 6

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).

BAF Complex Antibody Sampler Kit: Image 7

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).

BAF 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 SMARCC2/BAF170 (D8O9V) Rabbit mAb, 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. The figure shows binding across ESR1, a known target gene of SMARCC2/BAF170 (see additional figure containing ChIP-qPCR data).

BAF Complex Antibody Sampler Kit: Image 9

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.

BAF Complex Antibody Sampler Kit: Image 10

Immunohistochemical analysis of mouse colon using ARID1A/BAF250A (D2A8U) Rabbit mAb.

BAF Complex Antibody Sampler Kit: Image 11

Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either ARID1A/BAF250A (D2A8U) 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.

BAF Complex Antibody Sampler Kit: Image 12

Western blot analysis of extracts of various cell lines using Brg1 (A52) Antibody.

BAF Complex Antibody Sampler Kit: Image 13

Confocal immunofluorescent analysis of HeLa (positive, left) and NCCIT (negative, right) cells using BRM (D9E8B) XP® Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red).

BAF Complex Antibody Sampler Kit: Image 14

prostate:

Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using BRM (D9E8B) XP(R) Rabbit mAb.

BAF Complex Antibody Sampler Kit: Image 15

Western blot analysis of extracts from various cell lines using BRM (D9E8B) XP® Rabbit mAb (upper) or Brg1 (A52) Antibody #3508 (lower).

BAF Complex Antibody Sampler Kit: Image 16

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 SMARCB1/BAF47 (D8M1X) 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.

BAF 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, 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).

BAF Complex Antibody Sampler Kit: Image 18

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).

BAF Complex Antibody Sampler Kit: Image 19

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 SMARCC2/BAF170 (D8O9V) Rabbit mAb, 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. The figure shows binding across chromosome 6 (upper), including ESR1 (lower), a known target gene of SMARCC2/BAF170 (see additional figure containing ChIP-qPCR data).

BAF Complex Antibody Sampler Kit: Image 20

Immunohistochemical analysis of paraffin-embedded cell pellets, COS-7 (left) or T-47D (right), using ARID1A/BAF250A (D2A8U) Rabbit mAb.

BAF Complex Antibody Sampler Kit: Image 21

Immunohistochemical analysis of paraffin-embedded HeLa (left) and NCCIT (right) cell pellets using BRM (D9E8B) XP(R) Rabbit mAb.

BAF Complex Antibody Sampler Kit: Image 22

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using SMARCB1/BAF47 (D8M1X) Rabbit mAb.

BAF Complex Antibody Sampler Kit: Image 23

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.

BAF Complex Antibody Sampler Kit: Image 24

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.

BAF Complex Antibody Sampler Kit: Image 25

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 SMARCC2/BAF170 (D8O9V) 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.

BAF Complex Antibody Sampler Kit: Image 26

Immunohistochemical analysis of paraffin-embedded human lung adenosquamous carcinoma using ARID1A/BAF250A (D2A8U) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

BAF Complex Antibody Sampler Kit: Image 27

Immunohistochemical analysis of paraffin-embedded human clear cell adenocarcinoma of the ovary using BRM (D9E8B) XP(R) Rabbit mAb.

BAF Complex Antibody Sampler Kit: Image 28

Immunohistochemical analysis of paraffin-embedded mouse colon using SMARCB1/BAF47 (D8M1X) Rabbit mAb.

BAF Complex Antibody Sampler Kit: Image 29

Immunoprecipitation of SMARCC2/BAF170 from PANC-1 cell extracts, using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or SMARCC2/BAF170 (D8O9V) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using SMARCC2/BAF170 (D8O9V) Rabbit mAb.

BAF Complex Antibody Sampler Kit: Image 30

Western blot analysis of extracts from various cell lines using ARID1A/BAF250A (D2A8U) Rabbit mAb.

BAF Complex Antibody Sampler Kit: Image 31

Immunohistochemical analysis of paraffin-embedded endometrioid adenocarcinoma using SMARCB1/BAF47 (D8M1X) Rabbit mAb.

BAF Complex Antibody Sampler Kit: Image 32

Western blot analysis of extracts from various cell lines using SMARCC2/BAF170 (D8O9V) Rabbit mAb.

BAF Complex Antibody Sampler Kit: Image 33

Western blot analysis of T-47D and Jurkat cell extracts using ARID1A/BAF250A (D2A8U) Rabbit mAb (upper) or ß-Actin (D6A8) Rabbit mAb #8457 (lower). Additional ARID1A/BAF250A degradation products may be detected in some cell extracts between 135kDa-250kDa, which are absent in the ARID1A/BAF250A negative T-47D cell line.

BAF Complex Antibody Sampler Kit: Image 34

Immunohistochemical analysis of paraffin-embedded HeLa (left) and A204 (right) cell pellets using SMARCB1/BAF47 (D8M1X) Rabbit mAb.

BAF Complex Antibody Sampler Kit: Image 35

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using SMARCB1/BAF47 (D8M1X) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

BAF Complex Antibody Sampler Kit: Image 36

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.

BAF Complex Antibody Sampler Kit: Image 37

Immunoprecipitation of SMARCB1/BAF47 from 293T cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb XP® Isotype Control #3900, and lane 3 is SMARCB1/BAF47 (D8M1X) Rabbit mAb. Western blot analysis was performed using

SMARCB1/BAF47 (D8M1X) Rabbit mAb.

BAF Complex Antibody Sampler Kit: Image 38

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

BAF Complex Antibody Sampler Kit: Image 39

Western blot analysis of extracts from various cell lines using SMARCB1/BAF47 (D8M1X) Rabbit mab (upper) or β-Actin (13E5) Rabbit mAb #4970 (lower).

To Purchase # 12854T
Product # Size Price
12854T
1 Kit  (6 x 20 µl) $ 445

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
ARID1A/BAF250A (D2A8U) Rabbit mAb 12354 20 µl
  • WB
  • IHC
  • ChIP
H Mk M R 270 Rabbit IgG
Brg1 (A52) Antibody 3508 20 µl
  • WB
  • IF
H Mk M 220 Rabbit 
BRM (D9E8B) XP® Rabbit mAb 11966 20 µl
  • WB
  • IP
  • IHC
  • IF
  • ChIP
H Mk 200 Rabbit IgG
SMARCB1/BAF47 (D8M1X) Rabbit mAb 91735 20 µl
  • WB
  • IP
  • IHC
  • ChIP
H Mk M R 44 Rabbit IgG
SMARCC1/BAF155 (D7F8S) Rabbit mAb 11956 20 µl
  • WB
  • IP
  • ChIP
  • C&R
H Mk M R 155 Rabbit IgG
SMARCC2/BAF170 (D8O9V) Rabbit mAb 12760 20 µl
  • WB
  • IP
  • ChIP
H Mk M R 162, 170 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The BAF Complex Antibody Sampler Kit provides an economical means of detecting total protein from the SWI/SNF family members including ARID1A/BAF250A, Brg1, BRM, SMARCC1/BAF155, SMARCC2/BAF170 and SMARCB1/BAF47. The kit contains enough primary antibody to perform two western blots per primary antibody.

Specificity / Sensitivity

Each antibody in this kit recognizes endogenous levels of total protein for the specified target and does not cross-react with other family members. ARID1A/BAF250A (D2A8U) Rabbit mAb also cross-reacts with proteins of unknown origin at 65 kDa.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly1293 of human ARID1A/BAF250A protein, Gly264 of human BRM protein, Gly975 of human SMARCC1/BAF155 protein, Ile818 of human SMARCC2/BAF170 protein, or Leu120 of human SMARCB1/BAF47 protein. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala52 of human Brg1 protein. Polyclonal antibodies are purified by Protein A and peptide affinity chromatography.

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). BRM and BRG1 are also considered to be tumor suppressors and their expression levels are severely reduced in several cancer cell lines (10-13). SMARCC1/BAF155, SMARCC2/BAF170, and SMARCB1/BAF47 are members of the core subunits of the SWI/SNF complex, which is necessary for efficient nucleosome remodeling by BRG1 in vitro (14). ARID1A/BAF250A is one of the accessory subunits of the SWI/SNF complex (15). SMARCC1, SMARCB1, and ARID1A are an essential part of the mouse embryonic stem cell specific SWI/SNF complex (esBAF). SMARCC1 is necessary for early embryogenesis, especially proper brain and visceral endoderm development (16-18). SMARCB1 is necessary for early embryogenesis and hepatocyte differentiation (19,20). ARID1A is critical for ES cell pluripotency and differentiation into mesoderm-derived cardiomyocytes and adipocytes (15). While SMARCC2 has been shown to be part of the SWI/SNF complex in non-pluripotent cells, it is absent in pluripotent embryonic stem (ES) cells. Expression of SMARCC2 has been shown to be up-regulated in neurons/neuronal progenitors upon differentiation of mouse ES cells with retinoic acid, and exogenous expression of SMARCC2 leads to loss of stem cell pluripotency and self renewal (21).

  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. Yamamichi, N. et al. (2005) Oncogene 24, 5471-81.
  11. Reisman, D.N. et al. (2002) Oncogene 21, 1196-207.
  12. Shen, H. et al. (2008) Cancer Res 68, 10154-62.
  13. Weissman, B. and Knudsen, K.E. (2009) Cancer Res 69, 8223-30.
  14. Phelan, M.L. et al. (1999) Mol Cell 3, 247-53.
  15. Gao, X. et al. (2008) Proc Natl Acad Sci U S A 105, 6656-61.
  16. Han, D. et al. (2008) Dev Biol 315, 136-46.
  17. Kim, J.K. et al. (2001) Mol Cell Biol 21, 7787-95.
  18. Schaniel, C. et al. (2009) Stem Cells 27, 2979-91.
  19. Klochendler-Yeivin, A. et al. (2000) EMBO Rep 1, 500-6.
  20. Gresh, L. et al. (2005) EMBO J 24, 3313-24.
  21. Ho, L. et al. (2009) Proc Natl Acad Sci U S A 106, 5181-6.

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