GABPB1 Antibody

What is GABPB1 and why is it significant in research?

GABPB1 is a nuclear transcription factor that functions as part of the GA-binding protein complex, which regulates genes containing purine-rich GA repeats in their promoters. In humans, the canonical GABPB1 protein consists of 395 amino acid residues with a molecular mass of approximately 42.5 kDa . The protein exists in up to four different isoforms and is widely expressed across numerous tissue types .

GABPB1 is significant in research because it regulates genes involved in mitochondrial biogenesis, cell cycle progression, and cellular respiration. Recent studies have also implicated GABPB1 and its antisense transcript (GABPB1-AS1) in oxidative stress regulation , making it relevant to both basic cellular mechanisms and disease-related research.

What types of GABPB1 antibodies are available and how should researchers select the appropriate one?

GABPB1 antibodies are available in several formats, each with specific advantages for different applications:

When selecting a GABPB1 antibody, researchers should consider:

• The specific application (Western blot, IHC, etc.)

• Required species reactivity (human, mouse, rat)

• Whether specific isoform detection is necessary

• Validation data for the specific application

What are the optimal methods for validating GABPB1 antibody specificity?

Thorough validation is essential for ensuring experimental reproducibility. For GABPB1 antibodies, implement these validation approaches:

• Genetic validation: Use CRISPR knockout or siRNA knockdown of GABPB1 to confirm loss of signal.

• Peptide competition: Pre-incubate the antibody with its immunizing peptide to block specific binding.

• Molecular weight verification: Confirm detection at the expected molecular weight (approximately 42.5 kDa for canonical form) .

• Positive and negative controls: Use tissues/cells known to express or not express GABPB1. GABPB1 is widely expressed across many tissues, making it relatively accessible for positive controls .

• Multiple antibody comparison: Use antibodies recognizing different GABPB1 epitopes and compare staining patterns.

These validation steps should be documented in publications to enhance data reproducibility and reliability.

What are the optimal applications for GABPB1 antibodies in cellular and molecular research?

GABPB1 antibodies have been validated for multiple applications, with Western Blot and Immunohistochemistry being the most commonly reported :

For nuclear proteins like GABPB1, proper sample preparation is crucial. When performing Western blot, nuclear extraction protocols should be optimized to ensure efficient recovery of nuclear proteins .

How can researchers differentiate between GABPB1 isoforms using antibody-based techniques?

GABPB1 exists in up to four different isoforms , which may have distinct functional roles. To differentiate between isoforms:

• Epitope-specific antibodies: Select antibodies raised against peptides unique to specific isoforms. Consult immunogen information from manufacturers to identify which region of GABPB1 the antibody targets.

• Western blot optimization: Use gradient gels (4-15%) to achieve better separation of isoforms with similar molecular weights. Different isoforms can often be distinguished by their molecular weights:

  • Canonical GABPB1: 42.5 kDa

  • Other isoforms: May range from approximately 35-50 kDa

• 2D electrophoresis: Combine isoelectric focusing with SDS-PAGE to separate isoforms with similar molecular weights but different post-translational modifications.

• Validation approaches:

  • Use RT-PCR with isoform-specific primers in parallel with antibody detection

  • Implement isoform-specific knockdown to confirm band identity

  • Consider mass spectrometry validation following immunoprecipitation

When reporting results, clearly specify which isoform(s) are being detected and provide evidence supporting isoform identification.

What are the optimal protocols for studying GABPB1 in relation to oxidative stress pathways?

Recent research has linked GABPB1 and its antisense transcript GABPB1-AS1 to oxidative stress regulation . For studying this relationship:

• Oxidative stress induction protocols:

  • Chemical inducers: H₂O₂ (100-500 μM), paraquat (10-100 μM)

  • Time-course analysis (15 min to 24 hours post-induction)

  • Monitor ROS levels using fluorescent probes (DCFDA, CellROX)

• GABPB1 expression and localization analysis:

  • Nuclear/cytoplasmic fractionation to detect potential translocation

  • Time-dependent Western blot analysis with phospho-specific antibodies if available

  • Immunofluorescence to visualize subcellular localization changes

• Functional studies:

  • GABPB1 knockdown/overexpression followed by oxidative stress challenge

  • ChIP-seq before and after oxidative stress to identify differential binding

  • RT-qPCR of antioxidant response genes regulated by GABPB1

• GABPB1-AS1 co-regulation:

  • RNA-FISH combined with GABPB1 immunofluorescence to assess co-localization

  • Synchronized modulation of both GABPB1 and GABPB1-AS1 expression

  • RNA immunoprecipitation to detect potential direct interactions

These approaches enable mechanistic investigation of GABPB1's role in cellular responses to oxidative stress, with implications for various pathological conditions.

How should multiplexed detection of GABPB1 and interacting proteins be optimized?

Multiplexed detection enables visualization of GABPB1 in relation to other proteins in the same sample:

• Multiplex immunofluorescence optimization:

  • Select GABPB1 antibodies with demonstrated specificity

  • Choose antibodies raised in different host species to avoid cross-reactivity

  • Use fluorophores with minimal spectral overlap

  • Include nuclear counterstain (DAPI) in a non-overlapping channel

• Sequential staining protocol:

  • For panels with more than 4 antibodies, use sequential staining with tyramide signal amplification

  • Between rounds, perform complete antibody stripping with glycine buffer (pH 2.0)

  • Validate antibody removal between cycles

• Advanced multiplexing technologies:

  • Consider mass cytometry (CyTOF) for highly multiplexed protein detection

  • Imaging mass cytometry allows spatial visualization with up to 40 markers

  • Proximity ligation assay (PLA) can detect protein-protein interactions in situ

• Controls and analysis:

  • Include single-stained controls for spectral unmixing

  • Perform colocalization analysis with appropriate statistical measures

  • Use image analysis software for quantitative assessment of staining patterns

These approaches enable comprehensive analysis of GABPB1's molecular context within cells and tissues.

What approaches can resolve contradictory findings when using different GABPB1 antibodies?

Contradictory results with different antibodies are a common challenge. To resolve such discrepancies:

• Epitope mapping:

  • Determine the exact epitopes recognized by each antibody

  • Consider whether epitopes may be masked by protein interactions or modifications

  • Compare with protein domain structure and isoform-specific regions

• Validation hierarchy:

  • Establish genetic approaches (KO/KD) as validation gold standard

  • Compare antibody results against this standard

  • Rank antibodies based on specificity and consistency

• Multi-method verification:

  • Complement antibody-based methods with non-antibody techniques

  • Use mass spectrometry to corroborate protein identification

  • Implement RNA-level detection (RT-qPCR, RNA-seq) for correlation

• Standardized reporting:

  • Document complete antibody information (supplier, catalog number, lot, dilution)

  • Report all validation methods and results

  • Maintain consistent sample preparation protocols across experiments

By systematically addressing these factors, researchers can identify the source of discrepancies and establish more reliable protocols for GABPB1 detection.

How should researchers design optimal chromatin immunoprecipitation (ChIP) experiments for GABPB1?

As a transcription factor, GABPB1 binds to specific DNA sequences, making ChIP a valuable technique for studying its genomic targets:

• Cross-linking optimization:

  • Standard: 1% formaldehyde for 10 minutes at room temperature

  • For weaker interactions: Add protein-protein crosslinkers like DSG before formaldehyde

  • Quench with glycine (final concentration 0.125 M)

• Chromatin preparation:

  • Nuclear isolation to enrich for chromatin-bound proteins

  • Sonication to generate 200-500 bp DNA fragments

  • Verification of fragment size by agarose gel electrophoresis

• Immunoprecipitation conditions:

  • Pre-clearing with protein A/G beads and non-specific IgG

  • 3-5 μg GABPB1 antibody per reaction

  • Overnight incubation at 4°C with rotation

• Controls and validation:

  • Input DNA (5-10% of starting material)

  • IgG negative control (same species as GABPB1 antibody)

  • Positive control: qPCR for known GABPB1 target genes

  • Negative control: qPCR for regions without GA repeats

• Analysis approaches:

  • ChIP-qPCR for specific targets

  • ChIP-seq for genome-wide binding profile

  • Data presentation as percent input or fold enrichment over IgG

For optimal results, validate the ChIP-grade quality of GABPB1 antibodies before performing large-scale experiments or sequencing.

What are the critical considerations for quantifying GABPB1 expression levels?

Accurate quantification of GABPB1 requires attention to several methodological details:

• Western blot quantification:

  • Use recombinant GABPB1 protein standards for absolute quantification

  • Select appropriate loading controls (nuclear proteins like Lamin B1)

  • Implement linear dynamic range detection (fluorescent secondary antibodies)

  • Normalize to total protein using stain-free gels or Ponceau S

• Immunohistochemistry quantification:

  • Standardize image acquisition parameters

  • Use automated image analysis software for nuclear detection

  • Implement H-score or Allred scoring systems for semi-quantitative analysis

  • Include calibration standards on each slide for inter-slide normalization

• Flow cytometry quantification:

  • Use quantification beads for standardization

  • Implement robust gating strategies with FMO controls

  • Present data as molecules of equivalent soluble fluorochrome (MESF)

  • Standardize fixation and permeabilization protocols

• qPCR correlation:

  • Correlate protein levels with mRNA expression

  • Use multiple reference genes for normalization

  • Consider potential discrepancies due to post-transcriptional regulation

• Addressing variability:

  • Biological replicates (minimum n=3)

  • Technical replicates for each quantification method

  • Statistical analysis appropriate for sample size and distribution

These approaches ensure reliable quantification of GABPB1 expression across different experimental systems.

How can researchers account for post-translational modifications of GABPB1 in their experiments?

GABPB1 undergoes several post-translational modifications, including acetylation , which can affect function and antibody recognition:

• Modification-specific detection:

  • Use modification-specific antibodies when available (phospho-specific, acetyl-specific)

  • Validate specificity using recombinant proteins with defined modification status

  • Consider developing custom antibodies against known modification sites

• Modification manipulation:

  • Treat samples with phosphatases or deacetylases to remove modifications

  • Use inhibitors of modifying enzymes to enhance modification levels

  • Compare migration patterns before and after treatment

• Advanced detection methods:

  • Phos-tag gels for separating phosphorylated forms

  • 2D electrophoresis to separate proteins by charge and mass

  • Mass spectrometry following immunoprecipitation to map modification sites

• Functional correlation:

  • Correlate modification status with transcriptional activity

  • Design site-directed mutagenesis of modification sites

  • Monitor modification changes in response to cellular stimuli

These approaches enable researchers to understand how post-translational modifications regulate GABPB1 function in different cellular contexts.

How can researchers address weak or inconsistent GABPB1 signal in Western blots?

When GABPB1 detection yields weak or inconsistent signals, consider these optimization steps:

• Sample preparation optimization:

  • For nuclear proteins like GABPB1, ensure proper nuclear extraction

  • Add protease and phosphatase inhibitors immediately after lysis

  • Avoid multiple freeze-thaw cycles of protein samples

• Protein loading and transfer:

  • Increase protein loading (50-100 μg for nuclear extracts)

  • Verify transfer efficiency with reversible staining

  • For complete transfer of nuclear proteins, consider semi-dry transfer systems

• Antibody optimization:

  • Reduce primary antibody dilution (try 1:250 to 1:500)

  • Extend primary antibody incubation (overnight at 4°C)

  • Test different antibody clones targeting different epitopes

• Signal enhancement:

  • Use high-sensitivity ECL substrates

  • Implement biotin-streptavidin amplification systems

  • Consider fluorescent secondary antibodies for consistent quantification

• Blocking optimization:

  • Test different blocking agents (5% milk, 3-5% BSA)

  • Optimize blocking time (1-2 hours at room temperature)

  • Add 0.1% Tween-20 to reduce background while maintaining specific signal

If signal remains problematic, verify GABPB1 expression in your sample using RT-qPCR before concluding absence of the protein.

How should multiple bands in GABPB1 Western blots be interpreted?

Multiple bands in GABPB1 Western blots may represent isoforms, degradation products, or non-specific binding:

• Isoform identification:

  • Compare observed molecular weights with predicted isoform sizes

  • Canonical GABPB1: 42.5 kDa

  • Other isoforms may range from approximately 35-50 kDa

  • Up to 4 different isoforms have been reported

• Validation approaches:

  • Perform isoform-specific siRNA knockdown

  • Use isoform-specific antibodies when available

  • Compare patterns across different cell types with known expression profiles

• Post-translational modification assessment:

  • Treat samples with phosphatase or deacetylase

  • Look for band shifts indicating modification status

  • GABPB1 is known to undergo acetylation

• Sample preparation factors:

  • Test different lysis buffers to rule out extraction artifacts

  • Include reducing agent in sample buffer

  • Prepare fresh samples to minimize degradation

• Technical controls:

  • Run recombinant GABPB1 protein as size reference

  • Include positive control lysates with well-characterized band patterns

When reporting results, document all observed bands and provide interpretation based on validation studies.

What strategies can resolve non-specific background in GABPB1 immunostaining applications?

Non-specific background is a common challenge in immunostaining. For GABPB1 staining, implement these solutions:

• Blocking optimization:

  • Extend blocking time (1-2 hours)

  • Test different blocking agents (BSA, normal serum matching secondary antibody host)

  • Add 0.1-0.3% Triton X-100 for better antibody penetration

• Antibody dilution optimization:

  • Perform titration series to find optimal concentration

  • For nuclear antigens like GABPB1, higher dilutions often reduce background

  • Extend washing steps (at least 3x15 minutes between antibody incubations)

• Tissue-specific considerations:

  • Optimize fixation conditions (duration, temperature)

  • Test different antigen retrieval methods

  • Quench endogenous peroxidase activity for IHC

  • Treat with sodium borohydride to reduce autofluorescence for IF

• Controls:

  • Include primary antibody omission control

  • Use isotype control antibody

  • Perform peptide competition assay

These optimizations should yield clean nuclear staining for GABPB1 with minimal background, enabling accurate interpretation of expression patterns.

How can GABPB1 antibodies be integrated into single-cell analysis technologies?

Single-cell technologies provide unprecedented resolution for studying heterogeneous cell populations:

• Mass cytometry (CyTOF):

  • Metal-tagged GABPB1 antibodies enable multi-parameter analysis

  • Requires thorough validation and titration of metal-conjugated antibodies

  • Permits simultaneous detection of up to 40 proteins

• Single-cell Western blot:

  • Microfluidic platforms enable protein analysis at single-cell level

  • Requires optimization for low protein amounts

  • Can correlate GABPB1 with other transcription factors in the same cell

• Imaging mass cytometry:

  • Allows spatial visualization of GABPB1 in tissue context

  • Preserves tissue architecture while enabling highly multiplexed detection

  • Requires metal-conjugated antibodies with high specificity

• CITE-seq approaches:

  • Antibody-based protein detection combined with single-cell RNA-seq

  • Enables correlation between GABPB1 protein levels and transcriptome

  • Requires oligonucleotide-tagged antibodies

For all single-cell applications, thorough antibody validation is even more critical due to the limited material available for analysis and the inability to repeat experiments on the same cells.

What methodologies are optimal for studying the GABPB1/GABPB1-AS1 regulatory axis?

The relationship between GABPB1 and its antisense transcript GABPB1-AS1 represents an emerging research area :

• Co-detection methods:

  • RNA-FISH for GABPB1-AS1 combined with immunofluorescence for GABPB1

  • Sequential detection protocols with appropriate controls

  • Quantitative analysis of co-localization patterns

• Functional interrogation:

  • CRISPR/Cas9 targeting of either GABPB1 or GABPB1-AS1

  • Antisense oligonucleotides for specific knockdown

  • Overexpression systems with controlled induction

• Interaction analysis:

  • RNA immunoprecipitation (RIP) using GABPB1 antibodies

  • Chromatin isolation by RNA purification (ChIRP) for GABPB1-AS1

  • In vitro binding assays with recombinant proteins and synthesized RNA

• Oxidative stress modeling:

  • Treatments with oxidative stress inducers (H₂O₂, paraquat)

  • Monitoring of both GABPB1 protein and GABPB1-AS1 RNA levels

  • Assessment of downstream antioxidant response pathways

These approaches would provide valuable insights into the regulatory relationship between GABPB1 and its antisense transcript in normal physiology and under stress conditions.

How can advanced imaging techniques enhance GABPB1 protein interaction studies?

Advanced imaging approaches provide spatial information about GABPB1 interactions:

• Proximity ligation assay (PLA):

  • Detects protein-protein interactions with single-molecule sensitivity

  • Requires antibodies against GABPB1 and potential interaction partners

  • Provides spatial information about interaction sites within cells

  • Quantifiable by counting fluorescent spots

• FRET/FLIM analysis:

  • Measures direct protein-protein interactions based on energy transfer

  • Requires fluorescently tagged proteins or antibodies

  • Can detect transient interactions in living cells

  • Provides quantitative measurement of interaction efficiency

• Super-resolution microscopy:

  • Techniques like STORM, PALM, or SIM overcome diffraction limit

  • Enables visualization of protein clusters at nanoscale resolution

  • Can resolve nuclear distribution patterns of transcription factors

  • Requires highly specific antibodies with minimal background

• Live-cell imaging:

  • Fluorescent protein fusions to study dynamics

  • Optogenetic approaches to manipulate interactions

  • Complementation assays (BiFC) to visualize specific interactions

These techniques provide complementary information about GABPB1's molecular interactions and nuclear organization, helping to elucidate its role in transcriptional regulation.