PREB Antibody

What is PREB and what cellular functions should researchers consider when designing PREB antibody experiments?

PREB (Prolactin Regulatory Element Binding) is a 45 kDa protein that functions as a guanine nucleotide exchange factor (GEF) regulating the assembly of coat protein complex II/COPII in endoplasmic reticulum (ER) to Golgi vesicle-mediated transport . PREB selectively activates SAR1A and SAR1B by promoting the exchange of GDP for GTP in these small GTPases . In their activated state, these proteins insert into the ER membrane where they recruit the remainder of the COPII complex responsible for protein sorting and membrane vesicle formation .

PREB was initially identified for its regulatory role in pituitary gene transcription through binding to the prolactin gene (PRL) promoter, where it appears to activate transcription . When designing experiments, researchers should consider PREB's dual functionality in both vesicular transport and transcriptional regulation, as well as its predominantly ER localization.

How do expression patterns of PREB vary across tissues and cell lines for antibody validation purposes?

PREB shows differential expression across tissues and cell lines, which is crucial for designing proper validation controls:

Cell Line Expression (Validated in Western Blot):

Tissue Expression (Validated in IHC):

• Mouse intestine

• Human ovary tumor tissue

• Human prostate cancer tissue

• Human cerebral cortex (strong cytoplasmic positivity in neuronal cells)

• Human tonsil tissue

• Human liver (high expression predicted)

• Human placenta

This expression profile provides researchers with multiple options for positive controls when validating antibody specificity and optimizing experimental conditions.

What validated applications exist for PREB antibodies and what are the recommended protocol parameters?

PREB antibodies have been validated across multiple experimental applications with specific optimization parameters:

Western Blot (WB):

• Dilution ranges: 1:500-1:6000 (monoclonal) , 1:500-1:1000 (polyclonal) , 0.04-0.4 μg/ml

• Expected band: 45 kDa

• Recommended system: reducing conditions with appropriate buffer (e.g., Immunoblot Buffer Group 8)

• Example protocol: Use PVDF membrane, probe with antibody, follow with HRP-conjugated secondary antibody

Immunohistochemistry (IHC):

• Dilution ranges: 1:150-1:600 (monoclonal) , 1:500-1:2000 (polyclonal)

• Antigen retrieval: TE buffer pH 9.0 (recommended) or citrate buffer pH 6.0 (alternative)

• For paraffin sections: HIER pH 6 retrieval recommended

Immunofluorescence/Immunocytochemistry (IF/ICC):

• Dilution ranges: 1:50-1:500

• Fixation/permeabilization: PFA/Triton X-100

Immunoprecipitation (IP):

• Concentration: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Validated in: SKOV-3 cells

ELISA and Multiplex Assays:

• Matched antibody pairs available (e.g., 66981-2-PBS capture and 66981-3-PBS detection)

• Validated in: Cytometric bead array

Each application requires specific optimization, and researchers are advised to titrate the antibody in their specific testing system to achieve optimal results.

What are the recommended storage and handling conditions to maintain PREB antibody integrity and performance?

Proper storage and handling is crucial for maintaining antibody activity and reproducibility:

Storage Temperature:

• Most PREB antibodies: -20°C

• Conjugation-ready formats: -80°C

• Some formulations: -20 to -70°C

Buffer Composition:

• Standard format: PBS with 0.02% sodium azide and 50% glycerol pH 7.3

• Conjugation-ready format: PBS only (BSA and azide free)

• Alternative: PBS, pH 7.4, 150mM NaCl, 50% glycerol

Stability Period:

• Most formulations: Stable for one year after shipment

• R&D Systems formulation: 12 months from receipt, 1 month at 2-8°C after reconstitution, 6 months at -20 to -70°C after reconstitution

Handling Recommendations:

• Avoid repeated freeze-thaw cycles

• Aliquoting is unnecessary for -20°C storage in 50% glycerol formulations

• Small aliquots (20μl) may contain 0.1% BSA

• For long-term storage at 4°C, use small aliquots

Following these storage and handling guidelines will help maintain antibody performance and reproducibility across experiments.

What approaches should be used to validate PREB antibody specificity and what positive controls are recommended?

Rigorous validation is essential to ensure antibody specificity. Based on published methodologies:

Knockout/Knockdown Validation:

• Multiple studies have used KD/KO approaches for PREB antibody validation

• Comparison between control (vector-only) and PREB overexpression lysates creates an effective validation system

Recommended Positive Controls:

• Cell lines: SKOV-3, HepG2, or Raji cells show consistent PREB expression

• Tissues: Human tonsil tissue, human prostate cancer tissue

• Overexpression systems: HEK293T cells with PREB-DDK tag provide clear positives

Multiple Antibody Validation:

• Compare results with at least two different PREB antibodies recognizing distinct epitopes

• When possible, use both monoclonal and polyclonal antibodies targeting different regions of PREB

Specificity Testing Protocol Example:

• Prepare lysates from PREB-positive cells (e.g., HepG2) and PREB-knockdown cells

• Run Western blot with standardized protein loading (50 μg recommended)

• Probe with PREB antibody at optimized dilution

• Expected result: 45 kDa band in positive samples, reduced/absent in knockdown samples

This multi-faceted validation approach ensures antibody specificity and minimizes false positive/negative results.

How should researchers interpret variability in PREB antibody results between different detection methods?

Researchers may observe variations in PREB detection across different methods due to several factors:

Epitope Accessibility:

• In WB, denatured proteins expose all epitopes, while in IHC/IF, only surface-accessible epitopes are detected

• PREB contains two PQ-rich potential transactivation domains that may fold differently in native vs. denatured states

Method-Specific Considerations:

• Western Blot: Most reliable for molecular weight confirmation (consistently 45 kDa)

• Immunofluorescence: Typically shows ER localization pattern (endoplasmic reticulum)

• IHC: May show variable results depending on fixation and antigen retrieval methods

Reconciling Discrepancies:

• When discrepancies occur between methods, prioritize results from knockout/overexpression validation systems

• Consider that post-translational modifications may affect epitope recognition in specific applications

• Document fixation, antigen retrieval, and buffer conditions that affect reproducibility

Example Case Study:
In human A-431 cells, immunofluorescence showed consistent localization to the endoplasmic reticulum , while in cerebral cortex IHC, strong cytoplasmic positivity was observed specifically in neuronal cells . These differences reflect both methodology and tissue-specific expression patterns rather than antibody failure.

What is the documented cross-reactivity of PREB antibodies across species and what sequence homology considerations are important?

PREB is relatively conserved across mammalian species, but antibody reactivity varies:

Documented Reactivity:

Sequence Homology:

• Human PREB shares 87.7% amino acid sequence identity with both mouse and rat PREB

• Bovine cross-reactivity has been queried but not officially validated

Epitope Considerations:

• Antibodies raised against fusion proteins (e.g., PREB fusion protein Ag0202 ) may have different cross-reactivity profiles than those against specific peptide regions

• For monoclonal antibodies, the specific epitope location significantly impacts cross-species reactivity

Researchers working with non-human samples should perform preliminary validation when the specific species reactivity has not been documented, particularly when sequence divergence occurs in the epitope region.

How can PREB antibodies be effectively used in multiplex and high-throughput antibody applications?

PREB antibodies can be adapted for multiplex and high-throughput applications with specific optimization strategies:

Antibody Pair Selection:

• Validated antibody pairs like 66981-2-PBS (capture) and 66981-3-PBS (detection) have been optimized for cytometric bead arrays

• These conjugation-ready formats (BSA and azide-free in PBS) are specifically designed for multiplex applications

Conjugation Strategies:

• For fluorescent multiplex: Direct conjugation to fluorophores with minimal spectral overlap

• For bead-based assays: Biotinylation may be performed after buffer exchange to remove sodium azide

• Storage considerations: Small aliquots at -20°C post-conjugation

High-Throughput Optimization:

• For array/chip-based applications: Lower concentration ranges (0.04-0.1 μg/ml) often provide optimal signal-to-noise ratio

• Validation across multiple positive and negative control cell lines/tissues is essential to establish specificity in multiplexed systems

Co-IP Applications:

• PREB antibodies have been validated for co-immunoprecipitation studies

• When designing multiplex co-IP experiments, consider potential interaction partners involved in COPII vesicle formation or transcriptional regulation complexes

With appropriate optimization, PREB antibodies can be effectively incorporated into multiplex experimental designs while maintaining specificity and sensitivity.

What is known about PREB expression in disease contexts and how should antibody-based detection be optimized accordingly?

PREB has been studied in several disease contexts with specific detection considerations:

Cancer-Related Expression:

• Validated detection in human ovary tumor tissue

• Validated detection in human prostate cancer tissue

• Expression documented in various cancer cell lines including SKOV-3 (ovarian) , HepG2 (liver) , and A549 (lung)

Neurological Tissues:

• Strong cytoplasmic positivity observed in neuronal cells of human cerebral cortex

• Expression pattern differs from standard ER localization seen in cell lines

Method Optimization for Disease Models:

• For cancer tissues: TE buffer pH 9.0 recommended for antigen retrieval, with citrate buffer pH 6.0 as an alternative

• For neurological tissues: Specific fixation protocols may be required to preserve tissue architecture while maintaining epitope accessibility

• When comparing disease vs. normal tissues, standardize fixation, processing, and staining protocols to ensure comparable results

Quantitative Considerations:

• Semi-quantitative analysis of PREB expression should include multiple control tissues processed identically

• When differences in expression level rather than pattern are being assessed, validated internal loading controls are essential

Researchers studying PREB in disease contexts should validate antibodies specifically in their disease model of interest, as protein modifications or interacting partners may affect epitope accessibility.

How can PREB antibodies be integrated into high-resolution imaging techniques for subcellular localization studies?

PREB's function in ER-to-Golgi transport makes subcellular localization studies particularly relevant:

Super-Resolution Microscopy:

• For STED or STORM imaging: Higher primary antibody concentrations (1:50 dilution range) typically yield better results

• Secondary antibody selection: Highly cross-adsorbed secondary antibodies recommended to minimize background

• Co-localization studies with ER markers (e.g., calnexin) and COPII vesicle markers provide functional context

Sample Preparation Optimization:

• Fixation: 4% PFA recommended, avoid methanol which can disrupt membrane structures

• Permeabilization: Triton X-100 has been validated for PREB immunofluorescence

• Mounting medium: Use media with appropriate refractive index for the imaging technique

Validated Subcellular Localization:

• Endoplasmic reticulum localization has been confirmed in A-431 cells

• For co-localization studies, sequential staining may be necessary to avoid cross-reactivity

Quantitative Image Analysis:

• Pearson's correlation coefficient for co-localization with ER markers

• Distance measurements between PREB-positive structures and Golgi apparatus can provide insights into functional status

High-resolution imaging of PREB requires careful antibody validation, appropriate fixation and permeabilization protocols, and consideration of its dynamic localization during vesicular transport processes.

How do sequence variations and post-translational modifications affect PREB antibody epitope recognition?

Understanding sequence aspects of PREB is crucial for antibody selection and result interpretation:

Key Sequence Information:

• UniProt ID: Q9HCU5

• GenBank Accession Number: BC002765

• Gene ID (NCBI): 10113

• Full protein consists of 388 amino acids (Met1-Ser388)

Post-Translational Modifications:

• PREB contains PQ-rich domains that may undergo modifications affecting antibody binding

• Researchers should consider potential phosphorylation or other modifications when selecting antibodies for specific applications

Epitope Mapping Considerations:

• Antibodies targeting different regions may yield different results based on protein folding and accessibility

• For example, the E. coli-derived recombinant human PREB region K42-N236 is used as an immunogen for some antibodies

• Comparing results from antibodies recognizing different epitopes provides more comprehensive characterization

Cross-Reactivity Analysis:
When working with novel species or variant forms, researchers can use sequence alignment tools to predict epitope conservation and potential cross-reactivity based on the immunogen sequence region.

What recent advances in antibody validation and quality control should researchers consider when interpreting PREB antibody data?

Recent methodological advances have raised standards for antibody validation that should be applied to PREB studies:

Advanced Validation Approaches:

• Genetic strategies: Using CRISPR/Cas9-generated knockouts as gold-standard negative controls

• Independent antibody validation: Using multiple antibodies targeting different epitopes to confirm specificity

• Orthogonal validation: Correlating protein expression with mRNA expression data

• Expression validation: Testing across tissues and cell lines with varying expression levels

Quality Control Metrics:

• Measuring antibody batch-to-batch consistency through standardized reference samples

• Quantitative assessment of antibody specificity through immunoprecipitation-mass spectrometry

• Documentation of validation methods following minimal information about antibody characterization (MIAAB) guidelines

Recent Technological Advances:

• High-throughput sequencing of antibody repertoires allows unprecedented insight into antibody development and specificity

• Advanced recombinant antibody technologies are improving reproducibility compared to traditional hybridoma-derived antibodies

• Computational modeling of antibody-antigen interactions can predict potential cross-reactivity and epitope accessibility

Researchers should prioritize PREB antibodies with comprehensive validation documentation and consider implementing additional validation steps specific to their experimental system.

What are the most common problems encountered with PREB antibodies and what systematic troubleshooting approaches can resolve them?

When working with PREB antibodies, researchers may encounter several common issues:

In Western Blotting:

In Immunohistochemistry:

In Immunofluorescence:

Systematic troubleshooting using appropriate controls and standardized protocols will resolve most issues encountered with PREB antibodies.

How should researchers interpret discrepancies between published literature and their experimental results with PREB antibodies?

When experimental results differ from published findings, consider these analytical approaches:

Methodological Differences Analysis:

• Compare detailed protocols including antibody source, clone, dilution, incubation conditions, and detection systems

• Assess differences in sample preparation, particularly fixation methods and antigen retrieval

• Consider buffer compositions, especially for blocking and washing steps

Antibody Comparison:

• Different antibody clones target different epitopes, potentially yielding different results

• Monoclonal vs. polyclonal differences: monoclonals offer higher specificity but may be more sensitive to epitope modifications

• Commercial source variations: quality control standards vary between vendors

Biological Variability Factors:

• Cell line passage number and culture conditions can affect PREB expression

• Tissue source, preservation method, and processing can impact epitope accessibility

• Disease state, treatment conditions, or genetic background may alter PREB expression or localization

Resolution Strategies:

• Repeat experiments with multiple antibodies targeting different PREB epitopes

• Include appropriate positive and negative controls (especially knockout/knockdown systems)

• Quantify results using standardized methods and appropriate statistical analysis

• Document all methodological details to enable accurate comparison with published work

By systematically analyzing differences in methods, reagents, and biological systems, researchers can better understand and reconcile discrepancies with published literature.