srebf2 Antibody

What is SREBF2 and why is it significant in biological research?

SREBF2 (Sterol Regulatory Element Binding Transcription Factor 2) is a 1141-amino acid protein that functions as a master transcription factor primarily involved in cholesterol homeostasis. As a member of the SREBP family, it contains a basic helix-loop-helix (bHLH) domain and plays a critical role in regulating genes involved in cholesterol biosynthesis . SREBF2 is significant in research because:

• It functions as a transcriptional activator required for lipid homeostasis across multiple cell types

• Its expression is dynamically regulated in response to nutritional and hormonal cues

• Recent studies have implicated SREBF2 in immune cell regulation, particularly in tumor microenvironments

• Mutations in SREBF2 have been associated with hypercholesterolemia and altered glucose metabolism

What are the known isoforms of SREBF2 and how do they differ in detection using antibodies?

At least three isoforms of SREBF2 are known to exist, with researchers commonly observing two predominant forms in Western blot analysis :

• Full-length precursor form: Observed at approximately 120-130 kDa

• Cleaved/active form: Typically observed at 73-75 kDa

These different isoforms result from regulated proteolytic processing. When sterol concentrations are low, the SREBF2 precursor is transported from the endoplasmic reticulum to the Golgi apparatus where it undergoes proteolytic cleavage, releasing the transcriptionally active N-terminal domain that translocates to the nucleus . This processing must be considered when selecting antibodies for specific experimental applications, as some antibodies may preferentially detect one isoform over others depending on their epitope recognition sites .

How does SREBF2 differ from SREBF1, and how can I ensure antibody specificity between these family members?

While SREBF1 and SREBF2 belong to the same protein family and share structural similarities, they have distinct roles in metabolism:

• SREBF2 primarily regulates genes involved in cholesterol biosynthesis and uptake

• SREBF1 predominantly controls genes involved in fatty acid synthesis

To ensure antibody specificity:

• Select antibodies raised against non-conserved regions between SREBF1 and SREBF2

• Many commercial antibodies are specifically designed not to cross-react with SREBF1

• Validate specificity using knockout/knockdown controls or cells with known differential expression of these factors

• Consider using genetic approaches alongside antibody detection to confirm specificity

Research has shown that genetic deletion of TDP-43 in oligodendrocytes selectively targets the SREBF2 pathway while actually elevating SREBF1 expression, highlighting their distinct regulatory mechanisms and biological functions .

What are the optimal applications for different types of SREBF2 antibodies in research settings?

Based on current research applications, SREBF2 antibodies have been successfully employed in multiple techniques:

The choice of antibody should be guided by the specific research question and experimental system. For instance, certain applications like flow cytometry require cell permeabilization since SREBF2 is predominantly an intracellular target .

What is the optimal protocol for detecting both precursor and active forms of SREBF2 by Western blot?

For comprehensive detection of both SREBF2 isoforms by Western blot:

• Sample preparation:

  • Use RIPA buffer supplemented with protease inhibitors

  • For nuclear fraction enrichment (active form), perform subcellular fractionation

  • Include phosphatase inhibitors to preserve post-translational modifications

• Gel electrophoresis:

  • Use 5-10% gradient gels for optimal separation of both high (120-130 kDa) and low molecular weight (73-75 kDa) forms

  • Load 30-50 μg of total protein per well

• Transfer conditions:

  • Transfer at 150 mA for 50-90 minutes to nitrocellulose membrane

  • For the high molecular weight precursor form, extended transfer time may be necessary

• Blocking and antibody incubation:

  • Block with 5% non-fat milk/TBS for 1.5 hours at room temperature

  • Incubate with primary antibody (typically 0.5-2 μg/mL) overnight at 4°C

  • Wash with TBS-0.1% Tween 3 times (5 minutes each)

  • Incubate with appropriate HRP-conjugated secondary antibody (typically 1:5000 dilution)

• Detection:

  • Use enhanced chemiluminescence (ECL) detection

  • Expect bands at approximately 120-130 kDa (precursor) and 73-75 kDa (processed form)

This protocol has been validated in multiple cell types including THP-1, Jurkat, Raji, HL-60, as well as rat and mouse tissue samples .

How can I optimize immunofluorescence staining to distinguish between inactive and active SREBF2 in cells?

To differentiate between inactive (cytoplasmic/ER membrane-bound) and active (nuclear) forms of SREBF2 by immunofluorescence:

• Cell preparation:

  • For cultured cells: Grow cells on glass coverslips or chamber slides

  • For tissue sections: Use enzyme antigen retrieval (e.g., IHC enzyme antigen retrieval reagent) for 15 minutes

• Fixation and permeabilization:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1-0.5% Triton X-100 for 5-10 minutes

  • For membrane-bound precursor form: Gentler permeabilization with 0.05% saponin may preserve membrane structures

• Blocking:

  • Block with 10% normal goat serum (or serum matching secondary antibody host) for 1 hour

• Antibody incubation:

  • Primary antibody: Use 5 μg/mL anti-SREBF2 antibody overnight at 4°C

  • Secondary antibody: Fluorescent-conjugated secondary (e.g., DyLight 488) at 1:100 dilution for 30 minutes at 37°C

• Counterstaining and imaging:

  • Counterstain nuclei with DAPI

  • For colocalization studies: Include ER markers (e.g., calnexin) or Golgi markers (e.g., GM130)

  • Capture images using appropriate filter sets for the fluorophores used

• Analysis approach:

  • Active SREBF2: Predominantly nuclear localization

  • Inactive SREBF2: Perinuclear/reticular pattern consistent with ER localization

  • Quantify nuclear/cytoplasmic ratio to assess activation state

This approach has been validated in multiple cell lines including MCF-7 cells .

How can SREBF2 antibodies be used to investigate the relationship between cholesterol metabolism and immune function in the tumor microenvironment?

Recent research has uncovered important connections between SREBF2, cholesterol metabolism, and immune regulation in cancer contexts:

• Experimental design strategy:

  • Use SREBF2 antibodies in combination with CD63 (a marker for myeloid regulatory dendritic cells - mregDCs)

  • Perform multicolor flow cytometry to identify CD63+ mregDCs with elevated SREBF2 expression

  • Correlate SREBF2 activation with immunosuppressive phenotypes in tumor-draining lymph nodes

• Key findings from current research:

  • SREBF2-dependent gene program drives an immunotolerant CD63+ mregDC phenotype

  • Melanoma-derived lactate activates dendritic cell SREBF2 in the tumor microenvironment

  • These cells exhibit reduced capacity to drive CD8+ T cell proliferation while enhancing regulatory T cell differentiation

• Methodological approach:

  • Isolate CD63+ DCs from tumor-draining lymph nodes using FACS

  • Perform quantitative RT-PCR to measure expression of SREBF2 and its target genes

  • Validate using DC-specific genetic silencing and pharmacologic inhibition of SREBF2

  • Assess impact on anti-tumor CD8+ T cell activation and melanoma progression

This approach has revealed SREBF2 as a promising therapeutic target for overcoming immune tolerance in the tumor microenvironment, connecting metabolic regulation with immune function .

What techniques can be used to investigate SREBF2 binding to target gene promoters, and how do antibodies factor into these approaches?

To study SREBF2's role as a transcription factor binding to sterol regulatory elements (SREs):

• Chromatin Immunoprecipitation (ChIP):

  • Cross-link protein-DNA complexes in cells with formaldehyde

  • Lyse cells and shear chromatin by sonication to 200-500 bp fragments

  • Immunoprecipitate SREBF2-bound DNA using validated ChIP-grade SREBF2 antibodies

  • Reverse cross-links and purify DNA

  • Analyze by qPCR targeting known SREBF2-regulated promoters or by ChIP-seq for genome-wide binding analysis

• Electrophoretic Mobility Shift Assay (EMSA):

  • Design oligonucleotide probes containing SRE sequences

  • Prepare nuclear extracts from cells with active SREBF2

  • Incubate labeled probes with nuclear extracts

  • For supershift assays: Add SREBF2 antibody to confirm identity of DNA-binding protein

  • Analyze shifted complexes by non-denaturing gel electrophoresis

• Reporter gene assays:

  • Clone promoter regions containing SREs upstream of luciferase reporter

  • Co-transfect with SREBF2 expression constructs

  • For antibody-based inhibition: Microinject SREBF2 antibodies to block function

  • Measure luciferase activity to assess transcriptional activation

• CUT&RUN or CUT&Tag alternatives:

  • These newer techniques use antibody-directed targeting of micrococcal nuclease or Tn5 transposase

  • Require less starting material than traditional ChIP

  • Provide higher signal-to-noise ratio for transcription factor binding sites

  • Use validated SREBF2 antibodies conjugated to protein A/G

These approaches have been used to demonstrate that SREBF2 mutations can affect transcriptional activity and contribute to metabolic disorders like hypercholesterolemia .

How can researchers validate SREBF2 antibody specificity for critical experiments?

To ensure rigorous validation of SREBF2 antibodies for high-stakes experiments:

• Genetic validation:

  • Use CRISPR/Cas9 knockout or siRNA knockdown of SREBF2

  • Confirm absence or reduction of signal in Western blot, immunofluorescence, or flow cytometry

  • Example: Validate using SREBF2 knockdown/knockout in HepG2 or HeLa cells where SREBF2 is known to be expressed

• Peptide competition:

  • Pre-incubate antibody with excess immunizing peptide

  • Verify signal disappearance in intended application

  • Include non-competing peptide control

• Multi-antibody verification:

  • Use antibodies raised against different epitopes of SREBF2

  • Confirm consistent detection patterns across antibodies

  • Example: Compare antibodies targeting N-terminal vs. C-terminal regions to validate isoform detection

• Cross-reactivity assessment:

  • Test in samples with varied SREBF2 expression levels

  • Confirm absence of cross-reactivity with SREBF1 (a related family member)

  • Many commercial antibodies are specifically designed not to cross-react with SREBF1

• Multi-technique validation:

  • Compare protein detection across Western blot, immunofluorescence, and flow cytometry

  • Verify expected subcellular localization patterns

  • Correlate protein detection with mRNA expression using RNA-FISH or qPCR

Documented examples show that combining RNA-FISH with immunofluorescence can provide robust validation of SREBF2 antibody specificity and cellular localization .

Why might I detect unexpected molecular weight bands when using SREBF2 antibodies in Western blot?

Several factors can contribute to unexpected banding patterns when detecting SREBF2:

• Multiple isoforms:

  • Full-length precursor: Expected at 124 kDa (calculated)

  • Processed/mature form: Observed at 73-75 kDa

  • Intermediate processing products: May appear as bands between these sizes

• Post-translational modifications:

  • Phosphorylation can shift apparent molecular weight

  • SUMOylation or ubiquitination may produce higher molecular weight species

  • Glycosylation might affect mobility

• Proteolytic degradation:

  • Sample preparation without adequate protease inhibitors

  • Repeated freeze-thaw cycles

  • Extended storage at 4°C rather than -20°C

• Technical factors:

  • Incomplete denaturation: Use fresh sample buffer with SDS and heat to 95°C

  • Gel percentage: Use 5-10% gradient gels for better resolution

  • Transfer inefficiency: High molecular weight proteins may require longer transfer times

• Specific solutions:

  • Include multiple protease inhibitors in lysis buffer

  • Use freshly prepared samples when possible

  • Consider using phosphatase inhibitors if studying regulatory phosphorylation

  • Validate with multiple antibodies targeting different epitopes

Western blot analysis of SREBF2 in various cell lines has demonstrated successful detection of both major isoforms when appropriate conditions are used .

What are the most common pitfalls when using SREBF2 antibodies in immunofluorescence, and how can they be avoided?

Common challenges and solutions for SREBF2 immunofluorescence include:

• Low signal intensity:

  • Problem: Insufficient antibody concentration or epitope masking

  • Solution: Optimize antibody concentration (typically 5 μg/mL); try multiple antigen retrieval methods; increase incubation time to overnight at 4°C

• High background:

  • Problem: Non-specific binding or inadequate blocking

  • Solution: Increase blocking time (1-2 hours); use 10% normal serum from secondary antibody host species; include 0.1-0.3% Triton X-100 in blocking and antibody solutions

• Inconsistent nuclear localization:

  • Problem: Variable processing of SREBF2 due to culture conditions

  • Solution: Standardize cell culture conditions; consider cholesterol depletion to induce nuclear translocation; use subcellular markers to confirm localization

• Poor permeabilization:

  • Problem: Inadequate access to intracellular SREBF2

  • Solution: For fixed cells, use 0.1-0.5% Triton X-100 for 5-10 minutes; for flow cytometry, use specialized permeabilization buffers

• Artifactual localization:

  • Problem: Fixation-induced changes in protein localization

  • Solution: Compare different fixation methods (PFA vs. methanol); validate with live cell imaging using fluorescent protein-tagged SREBF2

• Validation approach:

  • Always include a negative control (secondary antibody only)

  • Use positive control tissues/cells with known SREBF2 expression

  • Consider SREBF2 knockdown controls for specificity verification

Published protocols have successfully used enzyme antigen retrieval for tissue sections and 4% paraformaldehyde fixation followed by permeabilization for cultured cells .

How can I optimize SREBF2 antibody-based detection in challenging samples like brain tissue where lipid content is high?

Brain tissue presents unique challenges for SREBF2 detection due to high lipid content and complex cellular architecture:

• Sample preparation considerations:

  • For fresh tissue: Rapid fixation to prevent degradation of transcription factors

  • For frozen sections: Optimal cutting temperature is critical (typically -20°C)

  • For fixed tissue: Extended fixation can mask epitopes, requiring aggressive retrieval

• Antigen retrieval optimization:

  • Heat-induced epitope retrieval: Citrate buffer (pH 6.0) at 95-100°C for 20 minutes

  • Enzymatic retrieval: For challenging tissues, enzyme digestion (proteinase K) can be effective

  • Combined approach: Heat followed by mild enzymatic treatment

• Detection enhancements:

  • Tyramide signal amplification for low-abundance targets

  • Extend primary antibody incubation to 48-72 hours at 4°C

  • Use specialized detergent mixtures to penetrate myelin-rich regions

• Background reduction strategies:

  • Pretreat sections with hydrogen peroxide to quench endogenous peroxidases

  • For fluorescence: Incubate with Sudan Black B (0.1-0.3%) to reduce lipofuscin autofluorescence

  • Use specialized blocking solutions containing fish gelatin or casein

• Validation approaches:

  • Combine protein detection with RNA-FISH for SREBF2 mRNA

  • Use genetic models with cell type-specific deletion of SREBF2 as negative controls

  • Compare multiple antibodies targeting different epitopes

Research has successfully used RNA-FISH combined with immunofluorescence to detect SREBF2 in oligodendrocytes in mouse spinal cord tissue, showing progressive reduction of SREBF2 in TDP-43-deleted oligodendrocytes .

How are SREBF2 antibodies being used to study metabolic disorders and potential therapeutic interventions?

SREBF2 antibodies are enabling several emerging research directions in metabolic disease:

• Genetic disorders of cholesterol metabolism:

  • Identification of SREBF2 mutations in patients with familial hypercholesterolemia

  • Characterization of mutation effects on protein function and localization

  • Assessment of variant SREBF2 response to statin therapy and other interventions

• Diabetes and glucose metabolism:

  • Investigation of SREBF2's role in pancreatic β-cell function

  • Analysis of SREBF2 activation in insulin-responsive tissues

  • Recent finding: SREBF2 mutations may contribute to combined hypercholesterolemia and hyperglycemia phenotypes

• Neurodegenerative diseases:

  • Study of SREBF2's role in maintaining myelin integrity in the nervous system

  • Investigation of cholesterol metabolism dysregulation in Alzheimer's disease

  • Analysis of TDP-43 regulation of SREBF2 in oligodendrocytes and its implications for amyotrophic lateral sclerosis

• Therapeutic development approaches:

  • Use of SREBF2 antibodies for target engagement studies in drug development

  • Screening compounds that modulate SREBF2 processing and activation

  • Validation of SREBF2 pathway inhibition in preclinical models

These applications employ multiple techniques including immunohistochemistry, Western blotting, ChIP, and reporter assays to characterize SREBF2 function and modulation in disease contexts .

What are the advantages and limitations of using SREBF2 antibodies for studying the relationship between lipid metabolism and cancer?

SREBF2 antibodies offer important insights into cancer metabolism research:

Advantages:

• Pathway activation assessment:

  • Direct measurement of SREBF2 nuclear translocation as a readout of pathway activation

  • Correlation with cholesterol synthesis gene expression in tumor samples

  • Identification of metabolic dependencies in specific cancer types

• Cell type-specific analysis:

  • Assessment of SREBF2 activity in tumor cells versus stromal/immune cells

  • Correlation with markers of cancer progression and metastasis

  • Investigation of metabolic crosstalk in the tumor microenvironment

• Therapeutic targeting validation:

  • Confirmation of on-target effects of drugs targeting the SREBF2 pathway

  • Biomarker development for patient stratification

  • Monitoring treatment response in preclinical models

Limitations:

• Technical challenges:

  • Heterogeneous tumor samples may yield variable results

  • Processing time can affect SREBF2 localization and detection

  • Limited sensitivity for detecting subtle changes in activation state

• Biological complexity:

  • SREBF2 activity is highly dynamic and responsive to multiple stimuli

  • Context-dependent regulation may differ between cancer types

  • Redundancy with SREBF1 in some metabolic pathways

• Therapeutic implications:

  • Systemic inhibition may affect normal tissues with high cholesterol requirements

  • Compensatory mechanisms may develop in response to SREBF2 inhibition

  • Challenges in developing specific inhibitors of transcription factor activity

Recent studies have leveraged SREBF2 antibodies to demonstrate that tumor-derived lactate activates SREBF2 in dendritic cells, promoting an immunosuppressive microenvironment, suggesting potential for combining metabolic and immunotherapeutic approaches .

How can SREBF2 antibodies be incorporated into high-throughput screening approaches for drug discovery?

Innovative applications of SREBF2 antibodies in drug discovery include:

• High-content imaging assays:

  • Automated immunofluorescence screening for compounds affecting SREBF2 nuclear translocation

  • Multiplexed detection with other pathway components (SCAP, Insig1/2)

  • Quantitative image analysis metrics: nuclear/cytoplasmic ratio, colocalization with ER/Golgi markers

  • Implementation: Fixed-cell format in 384/1536-well plates with automated liquid handling

• ELISA-based high-throughput screening:

  • Sandwich ELISA using capture and detection antibodies against different SREBF2 epitopes

  • Selective measurement of nuclear (processed) SREBF2 in fractionated samples

  • Adaptation to AlphaLISA or homogeneous time-resolved fluorescence (HTRF) formats for miniaturization

  • Application: Screening compound libraries for inhibitors of SREBF2 processing

• Reporter-based systems with antibody validation:

  • Luciferase reporters driven by SREBF2-responsive promoters (e.g., HMGCR, LDLR)

  • Confirmation of mechanism using SREBF2 antibodies in follow-up studies

  • Correlation of reporter activity with endogenous target gene expression

  • Advantage: Higher throughput with built-in functional readout

• Targeted protein degradation screening:

  • Assessing compounds that promote SREBF2 degradation rather than inhibiting processing

  • Western blot validation of total SREBF2 levels following compound treatment

  • Mechanistic studies using proteasome inhibitors and autophagy modulators

  • Potential: Development of SREBF2-directed PROTACs (proteolysis targeting chimeras)

• Validation strategies:

  • Orthogonal assays measuring cholesterol biosynthesis pathway activity

  • Genetic knockdown/knockout controls to confirm specificity

  • Structure-activity relationship studies correlated with SREBF2 inhibition patterns

These approaches have potential applications in developing therapeutics for hypercholesterolemia, cancer, and immune regulation .