BRG1 Antibody

What is BRG1 and what are its primary functions?

BRG1 (Brahma-related gene 1, also known as SMARCA4) is the catalytic ATPase subunit of the SWI/SNF chromatin remodeling complex. It functions primarily to:

• Alter DNA-nucleosome topology through ATP-dependent chromatin remodeling

• Participate in both transcriptional activation and repression

• Establish DNase I hypersensitivity sites characteristic of open chromatin

• Regulate nucleosome structure and positioning

• Mediate chromatin looping and higher-order chromatin organization

BRG1 is involved in the CREST-BRG1 complex, which regulates promoter activation by orchestrating calcium-dependent release of repressor complexes and recruitment of activator complexes. In resting neurons, BRG1 recruits phospho-RB1-HDAC repressor complexes to inhibit c-FOS promoter transcription, while calcium influx triggers changes enabling transcriptional activation .

BRG1 also belongs to neural progenitor-specific and neuron-specific chromatin remodeling complexes (npBAF and nBAF), playing crucial roles during neural development as cells transition from proliferating neural stem/progenitor cells to committed postmitotic neurons .

Rigorous validation approaches include:

• Genetic validation: Using BRG1/SMARCA4 knockout cell lines as negative controls. Multiple antibodies have been validated using HEK293T BRG1 knockout cell lines, showing complete absence of signal in knockout samples while detecting clear bands in parental lines .

• Knockdown validation: Using cells treated with BRG1-targeting siRNA or inducible shRNA systems. As described in one study: "To knockdown BRG1 expression in A1-A3 cells, cells were treated for 72 hr with Doxycycline" .

• Peptide competition: Pre-incubating the antibody with immunizing peptide to block specific binding. Some vendors provide blocking peptides for this purpose: "We do provide the blocking peptide for product anti-BRG1/SMARCA4 antibody (A00223-1)" .

• Cross-reactivity testing: Confirming the antibody doesn't recognize closely related proteins like BRM: "...using a BRG1 antibody that does not cross-react with BRM" .

• Multi-tissue validation: Testing antibody performance across different tissues with known BRG1 expression patterns, as demonstrated in one product validated in "human brain (cerebellum)" and "undifferentiated rat cortical stem cells" .

What is the expected molecular weight of BRG1 in Western blot?

BRG1 is consistently detected as a high molecular weight protein in Western blot applications:

• Typically appears at approximately 220-250 kDa under reducing conditions

• "A specific band was detected for Brg1 at approximately 220 kDa"

• "A specific band was detected for Brg1 at approximately 250 kDa"

The slight variation in observed molecular weight may be due to differences in:

• Post-translational modifications

• Gel systems and running conditions

• Protein standards used

• Cell/tissue type being analyzed

What tissues and cell types express BRG1?

According to the literature and antibody validation data, BRG1/SMARCA4 shows expression in:

• Brain tissues: "Brg1 was detected in immersion fixed paraffin-embedded sections of human brain (cerebellum)" and "fetal brain"

• Stem cells: "Brg1 was detected in immersion fixed undifferentiated rat cortical stem cells"

• Cancer cell lines: Including "HeLa human cervical epithelial carcinoma", "K562 human chronic myelogenous leukemia", "a549", "a431", "22rv1", and "u20s cells"

• Other tissues: "Tendon of biceps brachii, lung, cervix carcinoma, embryonic kidney, leukemic t-cell, erythroleukemia, liver, among other tissues"

BRG1 expression is primarily nuclear, with staining "localized to nuclei" as shown in IHC and IF applications .

How can BRG1 antibodies be used to study chromatin remodeling mechanisms?

BRG1 antibodies enable multiple advanced approaches to investigate chromatin remodeling:

• Genome-wide mapping of BRG1 binding sites using ChIP-seq:

  • High-depth sequencing (>60 million reads per ChIP-seq) can identify tens of thousands of BRG1 binding sites

  • "One hour of hormone exposure was sufficient to induce a massive DNA binding response by GR, with 29934 GR binding sites/peaks identified specifically in Dex-treated cells"

  • BRG1 binding patterns can be classified into distinct categories: "These classes exhibited distinct patterns of underlying chromatin accessibility, transcriptional activity, histone modification, and transcription factor motif enrichment and binding"

• Analysis of BRG1-mediated chromatin accessibility changes:

  • Combine BRG1 ChIP with DNase I hypersensitivity assays

  • "DNase I hypersensitivity sites at the MRE and α2 promoter are hypersensitive in wild-type FL nuclei, as expected, but are significantly less sensitive in mutant samples"

  • "Class I and Class III peaks gain chromatin accessibility upon Dex exposure and are associated with Dex-specific enhancer TSSs"

• Investigation of BRG1's impact on nucleosome positioning:

  • ChIP for histone H3 at BRG1 binding sites

  • "Compared to wild-type samples, mutants have increased total H3 at the MRE and α2 promoter"

  • This approach demonstrates how BRG1 influences nucleosome density at specific genomic loci

• Analysis of chromatin looping and higher-order structures:

  • "BRG1 directly regulates nucleosome structure and chromatin looping"

  • Combine BRG1 ChIP with chromosome conformation capture techniques (3C, 4C, Hi-C)

How should BRG1 antibodies be used in ChIP-seq experiments?

Based on published protocols, successful BRG1 ChIP-seq requires:

• Appropriate antibody selection:

  • Use antibodies validated specifically for ChIP applications

  • Consider epitope accessibility in crosslinked chromatin

• Optimized experimental design:

  • High sequencing depth: "We obtained data of high depth (>60 million reads per GR or BRG1 ChIP-seq)"

  • Stringent peak calling: "Called peaks using a 0.001 false discovery rate cutoff to ensure high confidence"

  • Appropriate controls: Include input chromatin and IgG controls

• Crosslinking and chromatin preparation:

  • Typically use 1% formaldehyde for 10-15 minutes at room temperature

  • Optimize sonication to achieve 200-500 bp chromatin fragments

• Data analysis considerations:

  • Compare BRG1 binding before and after stimuli to identify dynamic binding sites

  • Integrate with other genomic data: "BRG1 binding patterns at GR biding sites prior to and upon hormone signaling allowed us to define three classes of GR binding site"

  • Analyze overlap with transcription factors: "58% of GR peaks are overlapped by a BRG1 peak"

What is known about BRG1 interaction with other chromatin-associated proteins?

BRG1 forms complexes with multiple proteins involved in chromatin regulation:

• Interactions with SWI/SNF complex components:

  • BRG1 associates with BAFs (BRG1-associated factors)

  • "Mutant embryos also expressed normal levels of Brg1-associated factors (BAFs), which are noncatalytic subunits of SWI/SNF-related complexes"

  • "The Brg1 ENU1 protein was shown to physically interact with BAFs in coimmunoprecipitation assays"

• Interaction with Polycomb Repressive Complex 2 (PRC2):

  • "BRG1 has previously been shown to promote PRC2-mediated repression during stem cell lineage commitment"

  • "We found that EED was coimmunoprecipitated with endogenous BRG1, suggesting that BRG1 is present and able to form protein complexes with EED/PRC2"

  • "Endogenous BRG1 also co-immunoprecipitated with EED and H3K27me3 in primary mouse OPCs"

• Visualization of protein interactions using proximity ligation assay:

  • "We performed a proximity ligation assay (in situ PLA) to determine if BRG1 and EED physically interact"

  • "BRG1 and EED were detected in the same foci in the nuclei of primary OPCs"

• Interaction with glucocorticoid receptor:

  • "Further investigation showed that BRG1 controls how the glucocorticoid receptor affects the activity of genes"

  • "Our examination of the genomic glucocorticoid response demonstrates a previously undescribed role of BRG1 in patterning the underlying chromatin architecture"

How do specific BRG1 domains impact antibody selection for functional studies?

Strategic antibody selection based on BRG1 domains is critical for functional studies:

• ATPase domain studies:

  • The ATPase domain is critical for BRG1's remodeling function

  • A mutation study revealed "The mutant Brg1 protein is stable, assembles into SWI/SNF-related complexes, and exhibits normal ATPase activity but is unable to establish DNase I hypersensitivity sites characteristic of open chromatin"

  • For studying ATPase activity, antibodies targeting epitopes outside this domain are preferred

• Epitope accessibility in protein complexes:

  • BRG1 exists in large multi-protein complexes that may mask certain epitopes

  • Antibodies targeting exposed regions are more effective for immunoprecipitation

  • Different antibodies may be required depending on whether BRG1 is being studied in its free form or in complex

• Immunogen consideration:

  • The specific immunogen used to generate antibodies impacts their utility

  • Example immunogens include: "E. coli-derived human BRG1 recombinant protein (Position: Q555-E763)" and "E. coli-derived recombinant human Brg1 Gln673-Asn774"

  • Some antibodies are raised against synthetic peptides: "A synthetic peptide corresponding to a sequence within amino acids 200-300 of human BRG1/SMARCA4"

• Functionality-domain correlation:

  • Different domains mediate different functions (ATPase activity, protein-protein interactions, etc.)

  • For studying specific BRG1 functions, select antibodies targeting or avoiding relevant domains based on experimental goals

How can I investigate contradictory results when using BRG1 antibodies?

When faced with inconsistent results using BRG1 antibodies, consider these systematic troubleshooting approaches:

• Multiple antibody validation:

  • Use several antibodies targeting different BRG1 epitopes

  • Compare monoclonal vs. polyclonal antibody results

  • Vendors offer various formats: "Anti-BRG1/SMARCA4 Antibody Picoband" (rabbit) , "Human Brg1 Antibody" (rabbit monoclonal) , and "BRG1/SMARCA4 Rabbit Monoclonal Antibody"

• Evaluate potential technical variables:

  • Fixation impacts epitope accessibility: Different fixatives may mask certain epitopes

  • Buffer formulation: "If you require this antibody BSA free again in future, please do not hesitate to contact me"

  • Storage conditions affect antibody performance: "Store at -20˚C for one year from date of receipt. After reconstitution, at 4˚C for one month"

• Context-dependent expression analysis:

  • BRG1 may show tissue-specific expression patterns

  • "According to literature fetal brain does express SMARCA4"

  • "According to the expression profile of cervix carcinoma, SMARCA4 is highly expressed in cervix carcinoma"

• Genetic manipulation controls:

  • Include BRG1 knockout/knockdown samples

  • "To knockdown BRG1 expression in A1-A3 cells, cells were treated for 72 hr with Doxycycline"

• Post-translational modification consideration:

  • BRG1 undergoes various modifications that may affect antibody recognition

  • Phosphorylation states may alter epitope accessibility

What are optimal protocols for BRG1 antibody use in Western blotting?

Based on published methods, an optimized Western blot protocol for BRG1 detection includes:

• Sample preparation:

  • Use nuclear extracts when possible: "Gels were loaded with 30 µg of cytoplasmic (Cyto) and 15 µg of nuclear extracts (Nuc)"

  • Include protease inhibitors in lysis buffers: "Cell lysates were harvested in a lysis buffer containing protease and phosphatase inhibitors"

  • Use reducing conditions: "This experiment was conducted under reducing conditions"

• Gel selection and loading:

  • Use low percentage gels (6-8%) for optimal resolution of high molecular weight BRG1 (220-250 kDa)

  • Load appropriate amounts: 15-30 µg for nuclear extracts, more for whole cell lysates

• Transfer parameters:

  • Transfer to PVDF membrane: "PVDF membrane was probed with 0.5 µg/mL of Rabbit Anti-Human Brg1 Monoclonal Antibody"

  • Consider extended transfer times for the high molecular weight BRG1 protein

• Antibody incubation:

  • Recommended dilutions: 0.5-2 µg/mL

    • "PVDF membrane was probed with 0.5 µg/mL of Rabbit Anti-Human Brg1 Monoclonal Antibody"

    • "PVDF membrane was probed with 2 µg/mL of Goat Anti-Human Brg1 Antigen Affinity-purified Polyclonal Antibody"

  • Use appropriate buffer systems: "Using Immunoblot Buffer Group 1"

• Detection strategy:

  • Use HRP-conjugated secondary antibodies: "Followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody"

  • Include loading controls such as GAPDH: "GAPDH is shown as a loading control"

What co-immunoprecipitation protocols are effective for studying BRG1 protein interactions?

Effective Co-IP protocols for BRG1 include both endogenous and exogenous approaches:

• Endogenous Co-IP protocol:

  • "Primary mouse OPCs were cultured and then harvested in a lysis buffer containing protease and phosphatase inhibitors"

  • "Cell lysates were centrifuged at 13,000 rpm for 30 min at 4°C"

  • "Supernatants were incubated with 2 μg of IgG or anti-BRG1 antibody and immunoprecipitated using Protein A/G beads overnight at 4°C under rotation"

  • Include appropriate controls: "A mock immunoprecipitation was performed, where BRG1 antibody was omitted from wild-type embryo lysates, as a negative control"

• Exogenous Co-IP protocol (for tagged proteins):

  • "Oli-neu cells were transfected with HA-EED plasmid using Polyjet for 48 h"

  • "Cell lysates were centrifuged at 13,000 rpm for 30 min at 4°C"

  • "Supernatants were incubated with 20 μl of magnetic anti-HA beads overnight at 4°C under rotation"

• Antibody considerations:

  • Use antibodies validated for IP applications

  • Consider antibodies that don't cross-react with related proteins: "Using a BRG1 antibody that does not cross-react with BRM"

  • Typically use 2-5 μg of antibody per IP reaction

• Detection of interactions:

  • Western blot analysis of precipitated material

  • Mass spectrometry for unbiased identification of interacting partners

  • Confirmation with reverse Co-IP: immunoprecipitate the suspected partner and probe for BRG1

What are best practices for immunofluorescence and immunohistochemistry with BRG1 antibodies?

For optimal detection of BRG1 in tissues and cells:

• IHC on paraffin sections:

  • "Brg1 was detected in immersion fixed paraffin-embedded sections of human brain (cerebellum) using Rabbit Anti-Human Brg1 Monoclonal Antibody at 1 µg/mL for 1 hour at room temperature"

  • Detection systems: "Followed by incubation with the Anti-Rabbit IgG VisUCyte HRP Polymer Antibody"

  • Visualization: "Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue)"

  • Expected results: "Specific staining was localized to neuronal nuclei"

• Immunofluorescence on cultured cells:

  • "Brg1 was detected in immersion fixed undifferentiated rat cortical stem cells using Goat Anti-Human Brg1 Antigen Affinity-purified Polyclonal Antibody at 10 µg/mL for 3 hours at room temperature"

  • Detection: "Cells were stained using the NorthernLights 557-conjugated Anti-Goat IgG Secondary Antibody (red) and counterstained with DAPI (blue)"

  • Expected results: "Specific staining was localized to nuclei"

• Recommended antibody dilutions:

  • For IHC-P: 1-10 µg/mL or 1:50-1:200 dilution

  • For IF/ICC: 10 µg/mL or 1:50-1:200 dilution

• Antigen retrieval considerations:

  • Heat-induced epitope retrieval is typically necessary for formalin-fixed tissues

  • Buffer composition and pH should be optimized for BRG1 epitopes

How can I troubleshoot weak or non-specific signals when using BRG1 antibodies?

When facing detection challenges with BRG1 antibodies, consider these troubleshooting strategies:

• Weak signal solutions:

  • Increase antibody concentration: Test a range from manufacturer's recommendation up to 2-5x higher

  • Extend antibody incubation time: Overnight at 4°C instead of 1-3 hours

  • Optimize antigen retrieval: Try different buffers and heating methods

  • Use signal amplification systems: "Anti-Rabbit IgG VisUCyte HRP Polymer Antibody" provides enhanced sensitivity

  • For Western blots, use more sensitive detection reagents and longer exposure times

• Non-specific binding remedies:

  • Increase blocking stringency: Test different blocking agents (BSA, normal serum, commercial blockers)

  • Add detergents to antibody dilution buffers (0.1-0.3% Triton X-100)

  • Perform peptide competition: Some vendors provide blocking peptides

  • Pre-clear lysates in IP experiments: Incubate with beads alone before adding antibody

• Special considerations for nuclear proteins:

  • Ensure proper nuclear permeabilization for IF/ICC

  • For Western blots, use proper nuclear extraction protocols

  • "Gels were loaded with 30 µg of cytoplasmic (Cyto) and 15 µg of nuclear extracts (Nuc)"

• Sample preparation optimization:

  • Test different fixation protocols for IF/IHC

  • For Western blots, include phosphatase inhibitors and use fresh samples

  • For ChIP, optimize crosslinking conditions and sonication parameters

How do I select between monoclonal and polyclonal BRG1 antibodies for specific applications?

The choice between monoclonal and polyclonal BRG1 antibodies depends on experimental goals:

• Monoclonal antibody advantages:

  • Higher specificity for single epitope

  • "Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests"

  • Better for quantitative applications

  • Examples: "Anti-BRG1 antibody [EPR3912]" and "Anti-BRG1 antibody [EPNCIR111A]"

• Polyclonal antibody advantages:

  • Recognition of multiple epitopes may provide stronger signals

  • Better tolerance of minor epitope changes (fixation, denaturation)

  • May be advantageous for IP applications

  • Example: "Human Brg1 Antibody (Antigen Affinity-purified Polyclonal)"

• Application-specific considerations:

  • For Western blotting: Both types work well, monoclonals provide consistency

  • For IHC/IF: Polyclonals may provide stronger signals in fixed tissues

  • For ChIP: Monoclonals offer higher specificity for single epitopes

  • For therapeutic/diagnostic development: Monoclonals are preferred

• Special format considerations:

  • Some vendors offer enhanced performance formulations: "The brand Picoband indicates this is a premium antibody that guarantees superior quality, high affinity, and strong signals with minimal background"

  • Recombinant antibodies provide highest consistency: "Anti-BRG1 antibody [EPNCIR111A] - recombinant"