REEP1 Antibody

What is REEP1 and what is its cellular function?

REEP1 belongs to a family of six proteins originally identified for their ability to enhance heterologous expression of G protein-coupled receptors. At the cellular level, REEP1 has several critical functions:

• Required for endoplasmic reticulum (ER) network formation, shaping, and remodeling

• Links ER tubules to the cytoskeleton

• Facilitates ER-mitochondria interactions

• May enhance cell surface expression of odorant receptors

• Plays a role in long-term axonal maintenance

Research has demonstrated that REEP1 contains subdomains for both mitochondrial and ER localization and is detected in mitochondria-associated ER membranes (MAMs). Using a split-RLuc8 reassembly assay, researchers have shown that REEP1 facilitates ER-mitochondrial interactions in live cells, and that disease-associated mutations disrupt this function .

What is the tissue expression pattern of REEP1?

Unlike previous RT-PCR studies that suggested ubiquitous expression, immunoblotting analyses have demonstrated that REEP1 protein expression is restricted primarily to:

• Neuronal tissues (brain and spinal cord)

• Testes

This restricted expression pattern is consistent with the neurodegenerative phenotype observed in hereditary spastic paraplegia (HSP) and distal hereditary motor neuropathy type V (dHMN-V) caused by REEP1 mutations .

In cell culture studies, REEP1 expression was not detected in several non-neuronal cell lines tested, including HEK293, HEK293A, NRK, PC12, and Rat1 cells. When examining REEP1 expression in cultured murine sympathetic ganglion neurons, researchers found that REEP1 expression occurs between Day 4 and Day 8 of culture, with no expression detected at Day 1 or Day 4, but strong expression at Day 8 and Day 16 .

What are the standard applications for REEP1 antibodies in research?

REEP1 antibodies are employed in various research applications to study REEP1's role in normal cellular function and in neurodegenerative diseases:

Researchers have also used REEP1 antibodies in subcellular fractionation studies to determine REEP1's presence in different cellular compartments, particularly at the ER-mitochondria interface .

How can I validate the specificity of a REEP1 antibody?

Rigorous validation of REEP1 antibodies is essential for reliable research results. Effective validation approaches include:

• Immunoblotting with controlled samples:

  • Use tissues known to express REEP1 (brain, spinal cord) as positive controls

  • Use tissues/cells known not to express REEP1 as negative controls

  • Test against recombinant REEP1 protein

• Cross-reactivity testing:

  • Express tagged versions of different REEP family members (REEP1, REEP2, REEP6)

  • Verify the antibody only detects REEP1 and not other REEPs

• Knockout/knockdown validation:

  • Compare antibody reactivity in wild-type versus REEP1-depleted samples

• Immunofluorescence validation:

  • Compare staining patterns with known REEP1 localization

  • Perform co-localization studies with organelle markers

An example from the literature describes the validation of a REEP1 monoclonal antibody (mAb) clone N345/51, which was generated against a GST-fusion protein encoding amino acids #111-201 of mouse REEP1 carboxyl terminus. Its specificity was demonstrated by testing against Flag-REEP1, -REEP2, and -REEP6 transfected cells, confirming that it only detected Flag-REEP1 (calculated Mr = 23.4 kDa) .

How do REEP1 mutations contribute to hereditary spastic paraplegia?

REEP1 mutations are associated with hereditary spastic paraplegia (HSP), particularly the SPG31 form, accounting for approximately 6.5% of all HSP cases . These mutations lead to progressive lower-limb spastic paralysis through several mechanisms:

• Disruption of ER-mitochondria interactions:

  • REEP1 facilitates interaction between ER and mitochondria at MAMs

  • Disease-associated mutations diminish this function

  • These interactions are critical for calcium signaling, lipid transfer, and mitochondrial function

• Neuritic growth defects and degeneration:

  • Knockdown of REEP1 and expression of pathological REEP1 mutations result in neuritic growth defects and degeneration

  • These defects likely contribute to the axonal degeneration observed in HSP

• Mitochondrial dysfunction:

  • Impaired fusion/fission and defective bioenergetics have been reported in REEP1-associated HSP patients

  • REEP1 localizes to mitochondria and may directly impact mitochondrial function

Functional studies have provided the first evidence linking disrupted ER-mitochondria interactions to a failure in maintaining the health of long axons in HSPs, offering new avenues for potential therapeutic intervention .

What are the methodological approaches to resolve REEP1's cellular localization?

The cellular localization of REEP1 has been a subject of debate, with some studies suggesting ER localization and others indicating mitochondrial localization. Several methodological approaches have been used to resolve this conflict:

• Combined imaging and biochemical approaches:

  • Immunofluorescence microscopy with antibodies against REEP1 and organelle markers

  • Subcellular fractionation followed by Western blotting

  • Analysis of REEP1 in isolated mitochondria, ER, and MAM fractions

• Domain-specific studies:

  • Analysis of REEP1 subdomains for organelle targeting

  • Creation of deletion constructs to identify localization signals

• Co-localization analysis:

  • High-resolution microscopy to determine overlap with organelle markers

  • Quantitative analysis of co-localization coefficients

Using these approaches, researchers have demonstrated that REEP1 is present at the ER-mitochondria interface, containing subdomains for both mitochondrial and ER localization. Specifically:

• Endogenous REEP1 colocalizes with mitochondria in COS7 and MN-1 cells

• REEP1 is present in the mitochondrial but not the cytosolic cellular fraction by immunoblot

• Given its predicted transmembrane domains, REEP1 is likely a mitochondrial membrane protein

• REEP1 is detected in MAMs, which are contact sites between ER and mitochondria

These findings potentially reconcile the conflicting reports regarding REEP1 being either an ER or a mitochondrial protein .

How can I design experiments to study REEP1's role in ER-mitochondria interactions?

To investigate REEP1's role in ER-mitochondria interactions, consider the following experimental approaches:

• Split-RLuc8 reassembly assay:

  • A novel luciferase-based assay specifically developed for measuring ER-mitochondria interactions

  • Enables quantitative assessment of these interactions in live cells

  • Can detect changes caused by disease-associated mutations

• Subcellular fractionation and biochemical analysis:

  • Isolation of mitochondria-associated ER membranes (MAMs)

  • Western blotting with antibodies against:

    • REEP1 (rabbit polyclonal, 1:1000)

    • ATP5A (mouse monoclonal, 1:1000) for mitochondria

    • Calnexin (mouse monoclonal, 1:2000) for ER

    • Sigma-1R (rabbit polyclonal, 1:2000) for MAMs

• Microscopy approaches:

  • Co-localization studies with organelle markers

  • Live-cell imaging to track dynamic interactions

  • Super-resolution microscopy to better visualize contact sites

• Functional assays:

  • Calcium flux measurements between ER and mitochondria

  • Assessment of lipid transfer between organelles

  • Mitochondrial function tests (membrane potential, respiration)

• Genetic manipulation:

  • Expression of wild-type vs. mutant REEP1

  • Structure-function analysis using domain-specific constructs

  • CRISPR/Cas9-mediated introduction of disease-associated mutations

The split-RLuc8 assay has proven particularly valuable, showing that REEP1 facilitates ER-mitochondrial interactions in live cells and that this function is abrogated by disease-associated mutations .

How do REEP1 and REEP2 differ in their expression patterns and functions?

REEP1 and REEP2 are closely related members of the REEP family but exhibit distinct characteristics:

While both proteins are primarily expressed in neuronal tissues, REEP2 shows broader expression including tissues with neuronal-like exocytosis. Despite their sequence similarity, antibodies against REEP1 and REEP2 do not cross-react with each other .

Their functional differences are less well characterized, although REEP1's role in ER-mitochondria interactions and its connection to axonal maintenance in HSP has been established . The absence of disease associations for REEP2 suggests potentially divergent functional roles despite their structural similarities.

What controls are essential when studying REEP1 in neuronal cultures?

When studying REEP1 in neuronal cultures, comprehensive controls are crucial for reliable interpretation:

• Temporal expression controls:

  • Include neurons at different culture stages (Day 1, 4, 8, 16)

  • REEP1 expression begins between Day 4 and Day 8 in sympathetic ganglion neurons

  • Use early time points (Day 1, 4) as natural negative controls

• Antibody validation controls:

  • REEP1 knockout or knockdown neurons as negative controls

  • Secondary antibody-only controls for immunofluorescence

  • Multiple antibodies targeting different REEP1 epitopes when possible

• Genetic manipulation controls:

  • Empty vector or scrambled shRNA controls

  • Rescue experiments with wild-type REEP1 in knockdown neurons

  • Comparison of wild-type and disease-associated mutant REEP1

• Localization study controls:

  • Co-staining with organelle markers:

    • Mitochondrial markers (e.g., ATP5A)

    • ER markers (e.g., calnexin, calreticulin)

    • MAM markers (e.g., sigma-1R)

  • Quantitative co-localization analysis

• Functional assay controls:

  • Positive controls for neuritic growth or degeneration assays

  • Measurement of multiple morphological parameters

  • Inclusion of known modifiers of ER-mitochondria interactions

In published research, sympathetic ganglion neurons were analyzed at multiple time points using both RT-PCR and immunofluorescence staining to confirm REEP1's developmental expression pattern, with no expression at Day 1 or Day 4 but strong expression at Day 8 and Day 16 .

How can I differentiate between REEP family members in experimental systems?

Distinguishing between REEP family members requires careful experimental design:

• Antibody selection:

  • Choose antibodies raised against unique regions of specific REEP proteins

  • For REEP1, the carboxyl terminus (amino acids #111-201) has been used successfully for specific antibody generation

• Expression system validation:

  • Express tagged versions of different REEP family members

  • Perform immunoblotting with each antibody of interest

  • Verify each antibody only detects its intended target

• Molecular weight discrimination:

  • REEP1: ~22 kDa

  • REEP2: ~28 kDa

  • Use these size differences on Western blots

• Tissue expression patterns:

  • REEP1: Primarily neuronal tissues and testes

  • REEP2: Neuronal tissues plus pituitary and adrenal gland

  • Select tissues accordingly for positive/negative controls

• Genetic approaches:

  • Use isoform-specific siRNAs/shRNAs

  • CRISPR/Cas9 knockout of specific REEP family members

  • Rescue experiments with individual REEP proteins

In published research, investigators demonstrated that despite similar expression levels (confirmed with anti-Flag), a REEP1 monoclonal antibody only detected Flag-REEP1 and not Flag-REEP2 or Flag-REEP6. Similarly, REEP2 antisera only identified Flag-REEP2 and endogenous REEP2, without cross-reactivity to other REEPs .

What are optimal protocols for detecting REEP1 in brain tissue sections?

For immunohistochemical detection of REEP1 in brain tissue, the following methodological considerations are important:

• Tissue preparation:

  • Paraformaldehyde fixation (typically 4%)

  • Paraffin embedding or cryosectioning (10-20 μm sections)

• Antigen retrieval:

  • TE buffer pH 9.0 is recommended for many REEP1 antibodies

  • Alternative: Citrate buffer pH 6.0

• Antibody selection and dilution:

  • Use antibodies validated for IHC applications

  • Typical dilutions range from 1:50 to 1:500

  • Rabbit polyclonal antibodies have shown good results

• Detection systems:

  • Biotinylated secondary antibodies with avidin-biotin amplification

  • DAB chromogen for brightfield microscopy

  • Fluorescent secondary antibodies for immunofluorescence

• Controls:

  • Include REEP1-negative tissues

  • Primary antibody omission control

  • If possible, REEP1 knockout tissue as gold-standard negative control

The specific protocol should be optimized for each antibody and tissue processing method. Published studies have successfully detected REEP1 in paraffin-embedded human brain tissue using rabbit polyclonal antibodies at 1:100 dilution .

How can I resolve technical challenges in detecting REEP1 by Western blot?

Western blot detection of REEP1 can present specific technical challenges. Here are methodological approaches to address them:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for tissue lysis

  • For brain tissue, homogenize thoroughly to ensure complete extraction

  • Consider subcellular fractionation if studying organelle-specific distribution

• Protein loading and transfer:

  • Load adequate protein (typically 20-50 μg of total protein)

  • Use 12-15% gels to resolve the ~22 kDa REEP1 protein

  • Semi-dry or wet transfer protocols both work, but optimize transfer time for small proteins

• Antibody selection and blocking:

  • Primary antibody dilutions typically range from 1:500 to 1:2000

  • 5% non-fat dry milk in TBST is commonly used for blocking

  • Consider using BSA if background is problematic

• Detection optimization:

  • ECL kits: SuperSignal West Pico or Femto Chemiluminescent Substrate

  • Scanning systems: ChemiDoc™MP imaging system or equivalent

  • Longer exposure times may be needed for endogenous REEP1 detection

• Quantification:

  • Normalize REEP1 expression to housekeeping proteins

  • When comparing REEP1 mutants, normalize to expression tags (e.g., GFP)

• Troubleshooting:

  • No signal: Use positive control (brain tissue lysate)

  • Multiple bands: Validate specificity with knockout samples

  • High background: Optimize antibody dilution, blocking, and washing steps

Published protocols have successfully detected REEP1 using rabbit polyclonal antibodies (1:1000 dilution) and ECL-based development systems .

What methodologies are most effective for studying ER-mitochondria interactions involving REEP1?

For investigating REEP1's role in ER-mitochondria interactions, several complementary approaches have proven effective:

• The split-RLuc8 reassembly assay:

  • A novel luciferase-based system specifically developed for studying ER-mitochondria interactions

  • Allows quantitative assessment in live cells

  • Has successfully demonstrated REEP1's role in facilitating these interactions

  • Can detect how disease-associated mutations impact interaction strength

• Biochemical fractionation approaches:

  • Differential centrifugation to isolate subcellular compartments

  • Specific isolation of mitochondria-associated ER membranes (MAMs)

  • Western blotting with markers for:

    • Mitochondria: ATP5A (1:1000)

    • ER: Calnexin (1:2000)

    • MAMs: Sigma-1R (1:2000)

    • REEP1 (1:1000)

• Advanced microscopy techniques:

  • Confocal microscopy with organelle-specific markers

  • Live-cell imaging of fluorescently tagged REEP1 and organelles

  • Super-resolution microscopy to better visualize contact sites

  • Quantitative co-localization analysis

• Functional consequence assessment:

  • Calcium imaging to measure ER-mitochondria calcium transfer

  • Mitochondrial membrane potential measurements

  • Analysis of lipid transfer between organelles

• Structure-function analysis:

  • Expression of wild-type vs. disease-associated REEP1 mutants

  • Domain-specific constructs to identify regions critical for interactions

  • Rescue experiments in REEP1-depleted cells

The combination of these approaches has helped resolve the previously conflicting reports regarding REEP1 localization and has established REEP1's role in facilitating ER-mitochondria interactions, a function disrupted by disease-associated mutations .