REEP5 Antibody

What is REEP5 and why is it important in cardiovascular research?

REEP5 is a membrane protein belonging to the DP1/Yop1p protein family that regulates endoplasmic reticulum (ER) structure and function. It plays a critical role in cardiac health through several mechanisms:

• Acts as a regulator of sarco-endoplasmic reticulum (SR/ER) affecting cardiac functions

• Significantly enriched in both fetal and adult heart tissue despite broad expression across multiple tissues

• Downregulated in myocardial infarction and certain types of cardiomyopathy

• Mediates the function of CLEC5A to relieve myocardial infarction by inhibiting ER stress-induced apoptosis

REEP5 depletion can cause SR/ER membrane destabilization and luminal vacuolization, leading to decreased myocyte contractility and disrupted calcium handling, making it a promising target for cardiovascular disease intervention .

Verification of REEP5 antibody specificity is essential for ensuring reliable experimental results:

• Blocking experiments: Incubate your antibody with purified REEP5 protein (e.g., 10 μg of bacterially expressed, purified 6xHis-tagged REEP5) before immunostaining to confirm signal elimination

• REEP5 knockout controls: Use REEP5 knockout models (available through CRISPR/Cas9-based genome editing) as negative controls to confirm absence of signal

• Multiple antibody comparison: Use different REEP5 antibodies targeting distinct epitopes to confirm consistent localization patterns

• Western blot analysis: Verify the expected molecular weight (observed at 18-21 kDa, calculated at 21 kDa) and oligomerization patterns (monomers at 17 kDa, dimers at 34 kDa, and trimers at 51 kDa)

• Quantitative PCR validation: Confirm REEP5 mRNA levels correlate with protein expression patterns detected by antibody

What are the optimal sample preparation methods for REEP5 detection in cardiac tissue?

Proper sample preparation is critical for successful REEP5 detection in cardiac tissue:

For Western Blot Analysis:

• Use solubilization buffer containing 6M urea for optimal REEP5 extraction from membrane fractions

• Process samples fresh or flash-freeze immediately to prevent protein degradation

• Include protease inhibitors to prevent degradation of REEP5 protein

• Denature samples at 95°C for 5 minutes in SDS-loading buffer

For Immunohistochemistry:

• Fix cardiac tissues in methanol at room temperature for 5 minutes

• Permeabilize with 0.1% Triton X-100 for 15 minutes

• Block with 10% normal goat serum in PBS for 1 hour at room temperature

• For paraffin-embedded sections, perform antigen retrieval with TE buffer pH 9.0 or alternatively with citrate buffer pH 6.0

For Immunofluorescence of Cardiomyocytes:

• Allow dissociated cardiomyocytes to attach to laminin-coated glass coverslips before fixation

• Use primary antibody dilutions of 1:50 for REEP5

• Incubate with primary antibodies overnight at 4°C

How should I optimize REEP5 antibody concentrations for different experimental methods?

Optimization of REEP5 antibody concentrations depends on the specific application and antibody used:

Western Blot:

• Start with dilutions between 1:2000-1:10000 for polyclonal antibodies

• For monoclonal antibodies, higher dilutions (1:20000-1:100000) may be effective

• Test a dilution series to determine optimal signal-to-noise ratio

• Include positive controls (heart tissue) and negative controls (samples with REEP5 knockdown)

Immunohistochemistry:

• Begin with 1:300-1:1200 for polyclonal antibodies

• Use 1:5000-1:20000 for high-sensitivity monoclonal antibodies

• Perform titration experiments to optimize staining

• Include both positive control tissues (heart) and negative control tissues (tissues with low REEP5 expression)

Immunofluorescence:

• Start with 1:50-1:500 for polyclonal antibodies

• Use 1:400-1:1600 for monoclonal antibodies

• Always include a secondary antibody-only control to assess background

How can I simultaneously detect REEP5 and other cardiac proteins in co-localization studies?

Co-localization studies require careful planning to avoid antibody cross-reactivity and signal interference:

• Antibody selection considerations:

  • Choose REEP5 antibodies from different host species than your other target proteins

  • When using antibodies from the same host, use directly conjugated primary antibodies

  • Verify that secondary antibodies do not cross-react

• Recommended protocol:

  • Fix cardiomyocytes with methanol (5 minutes at room temperature)

  • Perform sequential staining with REEP5 antibody (1:50) and other target antibodies (e.g., RyR2 at 1:50)

  • Use species-specific secondary antibodies conjugated to spectrally distinct fluorophores (Alexa Fluor 488 and 555)

  • Include single-staining controls to assess bleed-through

  • Image using confocal microscopy for optimal resolution of subcellular structures

• Analysis approaches:

  • Calculate Pearson's or Mander's coefficients for quantitative co-localization assessment

  • Perform line scan analysis across structures of interest

  • Use deconvolution to improve resolution in densely packed structures

What are common issues when using REEP5 antibodies and how can they be resolved?

Researchers may encounter several challenges when working with REEP5 antibodies:

How do I accurately quantify changes in REEP5 expression in diseased versus normal cardiac tissue?

Accurate quantification of REEP5 expression changes requires multiple complementary approaches:

• Western blot quantification:

  • Use multiple biological and technical replicates

  • Normalize REEP5 band intensity to appropriate loading controls (β-tubulin, GAPDH)

  • Consider the different oligomeric forms when quantifying total REEP5 levels

  • Use validated quantification software (ImageJ, Image Studio Lite)

• Immunohistochemistry quantification:

  • Standardize image acquisition parameters

  • Take multiple random fields per section

  • Use automated image analysis software to quantify staining intensity and distribution

  • Perform blinded analysis to avoid bias

• qPCR for mRNA validation:

  • Use validated REEP5 primers spanning constitutively expressed exons

  • Normalize to multiple stable reference genes

  • Correlate mRNA changes with protein levels to identify post-transcriptional regulation

• Considerations for disease models:

  • REEP5 shows tissue-specific expression changes in different cardiovascular diseases

  • In myocardial infarction, REEP5 is downregulated in the infarct penumbra area

  • In pressure overload-induced heart failure, REEP5 expression is dynamic, showing initial decrease followed by later increase

  • In idiopathic cardiomyopathy, REEP5 levels may be elevated, while they are decreased in ischemic cardiomyopathy

How can I differentiate between specific REEP5 signal and non-specific binding in my experiments?

Differentiating specific from non-specific signals requires appropriate controls:

• Essential controls for validating specificity:

  • Blocking peptide control: Pre-incubate antibody with purified REEP5 protein to abolish specific signal

  • REEP5 knockdown/knockout: Use CRISPR/Cas9-edited cells or tissues with REEP5 depletion

  • Antibody isotype control: Use matched isotype control antibody at the same concentration

  • Secondary antibody only: Omit primary antibody to assess secondary antibody background

• Validation approaches:

  • Compare staining patterns using multiple REEP5 antibodies targeting different epitopes

  • Verify subcellular localization aligns with REEP5's known function as an ER membrane protein

  • Confirm molecular weight in Western blot matches predicted size (18-21 kDa)

  • Correlate protein detection with mRNA expression data

How can REEP5 antibodies be used to investigate the protein's role in ER stress and cardiac pathophysiology?

REEP5 antibodies enable sophisticated investigations into ER stress pathways:

• Subcellular co-localization studies:

  • Use REEP5 antibodies alongside markers for ER stress sensor proteins (PERK, IRE1α, ATF6)

  • Track translocation of ATF6 to the nucleus during ER stress with and without REEP5 overexpression

  • Visualize REEP5 interactions with CLEC5A using proximity ligation assays

• Biochemical interaction studies:

  • Perform co-immunoprecipitation with REEP5 antibodies to identify novel binding partners

  • Use REEP5 antibodies in chromatin immunoprecipitation to study transcriptional regulation

  • Combine with crosslinking approaches to capture transient interactions during ER stress response

• Functional studies in disease models:

  • Monitor REEP5 expression changes during progression of heart disease using Western blot and IHC

  • Correlate REEP5 levels with markers of ER stress (GRP78, phosphorylated PERK and IRE1α)

  • Assess impact of REEP5 manipulation on apoptotic markers (cleaved caspase-12, Chop)

  • Examine REEP5's role in calcium handling using simultaneous calcium imaging and immunofluorescence

What methodological approaches can resolve the conflicting REEP5 expression patterns reported in different cardiac disease models?

Resolving conflicting reports requires careful experimental design:

• Standardized disease model characterization:

  • Clearly define disease stage and severity using established markers (BNP for heart failure)

  • Document detailed methodology for creating disease models (e.g., LAD ligation, TAC, genetic models)

  • Measure functional parameters (EF, FS, LVESD, LVESV) to correlate with REEP5 expression

• Temporal profiling approaches:

  • Analyze REEP5 expression at multiple timepoints during disease progression

  • Consider the dynamic nature of REEP5 expression (initially decreased at 2-10 days post-TAC but increased after 21 days)

  • Use inducible expression systems to study acute versus chronic effects

• Regional analysis within cardiac tissue:

  • Examine region-specific differences (infarct core vs. penumbra vs. remote areas)

  • Use laser capture microdissection to isolate specific regions

  • Perform detailed immunohistochemical mapping across tissue sections

• Multi-level analysis:

  • Integrate transcriptomic (RNA-seq), proteomic, and functional data

  • Consider post-translational modifications and protein stability

  • Account for oligomerization states of REEP5 (monomers, dimers, trimers) which may vary by cardiac region

How can REEP5 antibodies be applied in cutting-edge cardiovascular single-cell research methodologies?

REEP5 antibodies can be integrated into advanced single-cell approaches:

• Single-cell protein profiling:

  • Use flow cytometry with REEP5 antibodies to characterize cellular heterogeneity in heart tissue

  • Apply Imaging Mass Cytometry (IMC) with metal-conjugated REEP5 antibodies for multiparameter analysis

  • Implement CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) to correlate REEP5 protein levels with transcriptome

• Spatial transcriptomics integration:

  • Combine REEP5 immunohistochemistry with spatial transcriptomics to map protein-mRNA relationships

  • Use multiplexed immunofluorescence to study REEP5 in relation to cell-type specific markers

  • Apply nearest-neighbor analysis to identify spatial relationships between REEP5-expressing cells and other cardiac cell populations

• Live cell imaging approaches:

  • Use cell-permeable, fluorescently-labeled REEP5 antibody fragments to track dynamic changes

  • Implement FRET-based biosensors to monitor REEP5 interactions with binding partners

  • Apply super-resolution microscopy techniques (STORM, PALM) to resolve REEP5 nano-organization within ER membranes

• Cancer research applications:

  • Examine REEP5's role in T-cell infiltration in esophageal squamous cell carcinoma using immunoprofiling

  • Investigate correlations between REEP5 and immune checkpoints (CTLA-4, TIM-3, HVEM) via multiplex IHC

  • Use single-cell clustering analysis to identify cell-specific expression patterns of REEP5 between cancerous and adjacent tissues

What emerging technologies might enhance the utility of REEP5 antibodies in cardiovascular research?

Several emerging technologies could significantly advance REEP5 research:

• Nanobody and recombinant antibody technologies:

  • Development of REEP5-specific nanobodies with improved tissue penetration

  • Creation of bispecific antibodies targeting REEP5 and binding partners simultaneously

  • Engineering of intrabodies for live-cell visualization of REEP5 dynamics

• Advanced imaging approaches:

  • Implementation of expansion microscopy to visualize REEP5 within complex ER networks

  • Application of correlative light and electron microscopy to study REEP5 at ultrastructural level

  • Development of light-sheet microscopy protocols for 3D visualization of REEP5 in whole-heart preparations

• Controlled protein modulation:

  • REEP5-targeting protein degradation systems (PROTAC, dTAG)

  • Optogenetic control of REEP5 function using antibody-guided photosensitizers

  • Antibody-drug conjugates for targeted delivery to REEP5-expressing tissues

How might REEP5 antibodies contribute to therapeutic development for cardiac diseases?

REEP5 antibodies could facilitate therapeutic development through several approaches:

• Target validation strategies:

  • Use antibodies to map REEP5 expression in patient samples

  • Correlate REEP5 levels with disease severity and treatment response

  • Identify patient subgroups that might benefit from REEP5-targeted therapies

• Therapeutic screening platforms:

  • Develop high-content screening assays using REEP5 antibodies to identify compounds that modulate REEP5 expression or localization

  • Create reporter systems to monitor ER stress responses in relation to REEP5 function

  • Screen for biologics that stabilize REEP5-mediated ER membrane organization

• Potential therapeutic mechanisms:

  • Target the REEP5-CLEC5A interaction to alleviate MI through inhibition of ER stress-induced apoptosis

  • Develop approaches to upregulate REEP5 in scenarios where its deficiency contributes to pathology

  • Engineer exosome-based delivery systems with REEP5-targeting capabilities

• Biomarker development:

  • Explore REEP5 as a potential biomarker for cardiovascular disease progression

  • Develop highly sensitive immunoassays for detecting REEP5 in circulation

  • Investigate REEP5 post-translational modifications as disease-specific indicators