SLC1A7 Antibody, HRP conjugated
Description
Definition and Basic Characteristics
SLC1A7 Antibody, HRP conjugated is a polyclonal immunoglobulin raised against specific epitopes of the human Excitatory Amino Acid Transporter 5 protein. The antibody specifically recognizes amino acid residues 115-220 of the SLC1A7 protein sequence, making it highly specific for detecting this particular glutamate transporter . This antibody is produced in rabbit hosts and is of the IgG isotype, ensuring appropriate specificity and binding capacity for research applications . The horseradish peroxidase (HRP) conjugation provides a direct enzymatic detection system that facilitates visualization in various experimental setups without requiring secondary antibody incubation steps . The antibody demonstrates high purity, typically >95%, achieved through Protein G purification methods that ensure minimal non-specific binding in experimental applications .
Physiological Functions
SLC1A7 (EAAT5) plays a crucial role in glutamate homeostasis, particularly in the retina where it is predominantly expressed. This transporter functions by removing L-glutamate from the synaptic cleft in a process that is both sodium- and voltage-dependent but chloride-independent . The protein's associated chloride conductance properties contribute significantly to visual processing mechanisms . SLC1A7 is strategically positioned near presynaptic release sites in photoreceptor ribbon synapses, where it plays a vital role in preventing glutamate excitotoxicity by efficiently clearing excess glutamate following synaptic transmission . This function is essential for maintaining proper signal transmission in retinal circuits and protecting retinal neurons from potential excitotoxic damage caused by excessive glutamatergic stimulation .
Expression Patterns
While SLC1A7/EAAT5 is predominantly expressed in retinal tissues, particularly in photoreceptor synapses, low levels of expression have also been detected in other tissues including liver, heart, muscle, and brain . The protein's expression pattern underscores its specialized role in glutamatergic signaling within the visual system. Within the retina, EAAT5 is specifically localized in close vicinity to presynaptic release sites of ribbon synapses, which are continuously active glutamatergic synapses containing presynaptic ribbons as structural specializations . This strategic localization enables EAAT5 to efficiently regulate glutamate concentrations at these highly active synapses, maintaining precise control over neurotransmission in the visual pathway . The protein's expression levels can be altered under pathological conditions, as demonstrated by studies showing reduced EAAT5 expression in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis .
Role in Glutamate Transport
SLC1A7/EAAT5 belongs to the sodium:dicarboxylate (SDF) symporter family and functions as a specialized glutamate transporter . The protein's primary role involves the uptake of L-glutamate from the extracellular space into cells, a process that requires sodium and is influenced by membrane voltage . This transport mechanism helps maintain appropriate glutamate concentrations at synapses, preventing the potential excitotoxic effects of excess glutamate accumulation. Additionally, SLC1A7 exhibits chloride conductance properties that contribute to visual processing, although this function is independent of its glutamate transport capability . The protein interacts with PDZ domains of DLG4 (also known as PSD-95), suggesting its involvement in protein-protein interaction networks that regulate synaptic organization and function . In photoreceptor synapses, EAAT5 works in concert with other components of the glutamate homeostasis machinery to ensure proper neurotransmission and prevent neuronal damage .
Application Areas
SLC1A7 Antibody, HRP conjugated has been validated primarily for Enzyme-Linked Immunosorbent Assay (ELISA) applications, making it particularly useful for quantitative detection of SLC1A7 protein in various sample types . While ELISA represents the most thoroughly validated application, related antibodies against the same target have demonstrated utility in other techniques including Western Blotting (WB), Immunohistochemistry (IHC), and Immunofluorescence (IF), suggesting potential broader applicability for the HRP-conjugated variant . The antibody's species reactivity is primarily focused on human samples, with potential cross-reactivity to other species requiring validation . The direct HRP conjugation eliminates the need for secondary antibody detection steps, streamlining experimental procedures and potentially reducing background signal in sensitive applications . This makes the antibody particularly valuable for high-throughput screening applications and diagnostic development where procedural efficiency is prioritized .
Common Research Applications
SLC1A7 Antibody, HRP conjugated serves as a valuable tool in numerous research applications focused on understanding glutamate transport mechanisms and retinal physiology. The antibody has been primarily validated for ELISA applications, where it enables quantitative detection of SLC1A7 protein in experimental samples with high sensitivity and specificity . In neuroscience research, this antibody facilitates investigations into glutamate transporter expression and localization in retinal tissues, contributing to our understanding of visual processing mechanisms and pathology . The antibody has potential applications in studies examining changes in SLC1A7/EAAT5 expression under various physiological and pathological conditions, including neuroinflammatory disorders like multiple sclerosis . Its specificity for human SLC1A7 makes it particularly valuable for translational research aiming to connect findings from animal models to human disease mechanisms . Additionally, the direct HRP conjugation enables streamlined detection protocols in immunohistochemical applications, potentially allowing for visualization of SLC1A7 distribution in tissue sections from normal and diseased states .
Experimental Protocols
When utilizing SLC1A7 Antibody, HRP conjugated for experimental applications, specific protocols must be followed to ensure optimal results. For ELISA applications, the antibody is typically used at dilutions determined empirically for each specific assay setup, with recommended starting concentrations often provided by manufacturers . The general workflow involves coating plates with capture antibody or antigen, blocking non-specific binding sites, incubating with samples containing the target protein, and detecting with the HRP-conjugated SLC1A7 antibody . For potential immunofluorescence applications, protocols typically involve tissue fixation, permeabilization, blocking, and overnight incubation with the antibody at 4°C, followed by visualization using appropriate HRP substrates . When used for Western blotting, samples are typically separated by SDS-PAGE, transferred to membranes, blocked, and probed with the HRP-conjugated antibody, followed by chemiluminescent detection . Specificity controls are essential in all applications and may include pre-absorption of the antibody with the immunizing peptide to confirm signal specificity, as demonstrated in studies examining EAAT5 expression in retinal tissues .
Quality Control and Validation
Rigorous quality control and validation procedures ensure the reliability of SLC1A7 Antibody, HRP conjugated for research applications. Manufacturers typically validate antibody specificity through various techniques, including ELISA against the immunizing peptide and potentially Western blot analysis of target tissues known to express SLC1A7 . Purity assessments confirm that the antibody preparation exceeds 95% purity through Protein G purification methods, minimizing the presence of contaminants that could contribute to non-specific binding . Functional validation includes testing the conjugated HRP enzyme activity to ensure proper signal generation in detection systems . Research studies utilizing similar antibodies have employed additional validation approaches, such as pre-absorption experiments where the antibody is incubated with excess target antigen to confirm binding specificity . The experimental use of control samples lacking SLC1A7 expression provides further confirmation of antibody specificity in research contexts . These validation steps collectively ensure that signals detected with the SLC1A7 Antibody, HRP conjugated genuinely represent the presence and distribution of the target protein in experimental samples .
Role in Disease Mechanisms
Research utilizing SLC1A7/EAAT5 antibodies has revealed important insights into disease mechanisms, particularly in neurological disorders affecting the visual system. In experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, studies have demonstrated significantly reduced EAAT5 expression at photoreceptor synapses as early as day 9 post-immunization . This finding suggests that alterations in glutamate transporter function may contribute to the pathogenesis of multiple sclerosis by disrupting glutamate homeostasis in the retina . The decreased expression of EAAT5 observed in these models potentially contributes to excitotoxic damage in retinal neurons due to impaired glutamate clearance from synaptic sites . Given that SLC1A7/EAAT5 plays a crucial role in maintaining appropriate glutamate concentrations at photoreceptor synapses, its dysfunction may lead to aberrant neurotransmission and potential neurodegeneration in the visual pathway . These observations highlight the importance of glutamate transporters as potential contributors to neuroinflammatory and neurodegenerative processes, extending beyond their conventional roles in normal neurotransmission .
Potential Therapeutic Implications
The identification of altered SLC1A7/EAAT5 expression in disease models opens avenues for potential therapeutic interventions targeting glutamate transport mechanisms. Strategies aimed at preserving or enhancing EAAT5 function could potentially mitigate excitotoxic damage in conditions characterized by glutamate dysregulation, such as multiple sclerosis with visual system involvement . The availability of specific antibodies like SLC1A7 Antibody, HRP conjugated enables precise monitoring of transporter expression levels in response to therapeutic interventions, facilitating the development and validation of targeted treatments . Furthermore, understanding the regulatory mechanisms controlling SLC1A7 expression could lead to the identification of molecular pathways amenable to pharmacological modulation, potentially preserving glutamate transporter function in pathological conditions . The specificity of EAAT5 expression in retinal tissues also suggests the possibility of developing targeted therapies that selectively affect visual system glutamate handling without disrupting glutamatergic signaling throughout the central nervous system . While direct therapeutic applications remain in the exploratory phase, the fundamental insights gained through research utilizing SLC1A7 antibodies contribute significantly to our understanding of disease mechanisms and potential intervention strategies .
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
What is SLC1A7 and why is it significant in research?
SLC1A7, also known as Excitatory Amino Acid Transporter 5 (EAAT5), was initially identified as a glutamate transporter coupled to chloride conductance primarily expressed in the retina. More recent evidence indicates it is also widely expressed in peripheral tissues. SLC1A7 functions as a low-affinity/low-capacity glutamate transporter with an anion channel optimized for conductance in the negative voltage range . Its study is significant for understanding glutamate signaling and transport mechanisms in both neural and non-neural tissues.
What applications are SLC1A7 HRP-conjugated antibodies suitable for?
SLC1A7 HRP-conjugated antibodies are primarily suitable for ELISA applications but can also be used in immunohistochemistry (IHC), western blotting (WB), and immunofluorescence (IF) depending on the specific antibody formulation . The HRP conjugation eliminates the need for secondary antibody incubation, allowing for more direct detection methods in these applications.
What is the expected molecular weight for SLC1A7 in western blotting?
The observed molecular weight for SLC1A7 in western blotting is approximately 68 kDa, while the calculated molecular weight is approximately 60.7 kDa . This discrepancy is likely due to post-translational modifications such as glycosylation, which is common in membrane transport proteins.
What species reactivity is available for SLC1A7 HRP-conjugated antibodies?
Commercial SLC1A7 HRP-conjugated antibodies typically show reactivity with human samples, and some formulations also cross-react with rat samples . For example, the antibody cataloged as ABIN7152247 shows reactivity with human samples, while other formulations (like A09067) may show reactivity with both human and mouse samples .
How can I validate the specificity of SLC1A7 HRP-conjugated antibodies for my particular tissue of interest?
Validation of SLC1A7 antibodies requires a multi-faceted approach, especially when investigating tissues with potentially low expression levels. Begin with positive controls from tissues known to express SLC1A7 (retina is ideal). For enhanced validation, utilize blocking peptides corresponding to the immunogen (typically AA 115-220 for many commercial antibodies) . Compare staining patterns with published literature, and consider knockdown/knockout validation if working with cell lines. For tissues not previously characterized, validate using multiple antibodies targeting different epitopes of SLC1A7 and complementary techniques such as RNA-seq or RT-PCR.
How does the binding specificity of different SLC1A7 antibodies affect experimental outcomes?
Different commercial SLC1A7 antibodies target distinct epitopes, which can significantly impact experimental results. Antibodies targeting AA 115-220 will recognize different conformational states compared to those targeting the C-terminus (AA 460-510) . This distinction becomes crucial when studying protein-protein interactions, conformational changes during transport, or when certain epitopes might be masked by protein folding or complex formation. When discrepancies in results occur between different antibodies, epitope availability should be considered as a potential explanatory factor.
What are the optimization strategies for using SLC1A7 HRP-conjugated antibodies in multiplex immunofluorescence studies?
For multiplex studies incorporating SLC1A7 HRP-conjugated antibodies:
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Sequential detection approach: Convert HRP signal to a fluorescent signal using tyramide signal amplification (TSA)
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Careful antibody panel design to avoid spectral overlap
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Optimization of antibody concentration (typically 1:100 to 1:500 dilution)
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Extended incubation times (overnight at 4°C) for enhanced signal-to-noise ratio
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Implementation of stringent washing steps to minimize background
This approach allows for simultaneous visualization of SLC1A7 with other markers of interest in complex tissue architecture .
How can I distinguish between SLC1A7 and other glutamate transporters when studying neural tissue?
Distinguishing SLC1A7 from other glutamate transporters requires careful experimental design. The table below summarizes the key differences:
| Transporter | Molecular Weight | Expression Pattern | Transport Properties | Antibody Epitope Selection |
|---|---|---|---|---|
| SLC1A7 (EAAT5) | 68 kDa | Primarily retina | Low-affinity/low-capacity | AA 115-220 or C-terminal |
| SLC1A1 (EAAT3) | 57 kDa | Neurons | High-affinity | Distinct N-terminal |
| SLC1A2 (EAAT2) | 62 kDa | Astrocytes | High-capacity | Distinct C-terminal |
| SLC1A3 (EAAT1) | 59 kDa | Astrocytes | High-affinity | Mid-region |
Select antibodies targeting unique regions and validate using tissues with differential expression of these transporters. For definitive discrimination, consider functional assays that exploit the distinct transport properties of SLC1A7, particularly its chloride conductance characteristics .
What are the optimal fixation and antigen retrieval methods when using SLC1A7 HRP-conjugated antibodies for immunohistochemistry?
For optimal results with SLC1A7 HRP-conjugated antibodies in immunohistochemistry:
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Fixation: 4% paraformaldehyde for 24-48 hours provides optimal epitope preservation
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Antigen retrieval: Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) at 95-98°C for 20 minutes
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Blocking: 5-10% normal serum (matching secondary antibody host if using unconjugated primary) with 0.1-0.3% Triton X-100
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Antibody dilution: Typically 1:100 to 1:500 in blocking buffer
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Incubation: Overnight at 4°C for primary antibody
These parameters should be optimized for each tissue type, as membrane proteins like SLC1A7 can be particularly sensitive to overfixation .
How can I quantitatively assess SLC1A7 expression levels using HRP-conjugated antibodies?
Quantitative assessment of SLC1A7 expression using HRP-conjugated antibodies can be achieved through:
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ELISA: Develop a standard curve using recombinant SLC1A7 protein (115-220AA)
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Western blotting: Densitometric analysis with normalization to housekeeping proteins
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Immunohistochemistry: Digital image analysis with appropriate controls:
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Positive control (retinal tissue)
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Negative control (antibody diluent only)
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Isotype control (matched IgG)
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Absorption control (pre-incubation with immunizing peptide)
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For all methods, include a range of known concentrations of recombinant SLC1A7 to generate standard curves. Report results as relative expression or concentration per unit protein .
What are the considerations when designing knockout validation experiments for SLC1A7 antibodies?
When designing knockout validation experiments:
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Select appropriate gene editing approach: CRISPR-Cas9 targeting conserved exons is preferred
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Target specific regions: Exons encoding AA 115-220 (common antibody target region)
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Confirm knockout at:
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Genomic level (PCR and sequencing)
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Transcript level (RT-PCR)
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Protein level (western blot with multiple antibodies)
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Include functional assays (glutamate transport activity)
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Test antibody in paired wild-type and knockout samples
Be aware that complete knockout may be embryonically lethal or cause compensatory upregulation of other transporters, complicating interpretation .
How can competitive enzyme immunoassay techniques be optimized for SLC1A7 detection?
For optimizing competitive enzyme immunoassays with SLC1A7 HRP-conjugated antibodies:
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Buffer optimization: PBS with 0.1% BSA and 0.05% Tween-20 typically provides optimal signal-to-noise ratio
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Sample preparation: Membrane protein extraction using mild detergents (0.5% NP-40 or 0.1% Triton X-100)
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Standard curve range: 0.1-100 ng/mL of recombinant SLC1A7
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Incubation conditions: 1-2 hours at room temperature or overnight at 4°C
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Washing protocol: 5 washes with PBS-T (0.05% Tween-20)
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Substrate reaction: TMB substrate with monitoring at 450nm
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Data analysis: Four-parameter logistic curve fitting
This approach allows for sensitive detection of SLC1A7 from complex biological samples, with typical assay sensitivity in the range of 0.1-1 ng/mL .
How should I address inconsistent results between different applications using SLC1A7 HRP-conjugated antibodies?
When facing inconsistencies between applications (e.g., positive ELISA but negative western blot):
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Consider epitope availability: The AA 115-220 region may be exposed differently in native versus denatured states
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Evaluate buffer compatibility: HRP conjugation may be sensitive to reducing agents or detergents
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Examine protein extraction methods: Membrane proteins require specialized extraction protocols
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Adjust antibody concentration: HRP-conjugated antibodies often require higher concentrations than unconjugated antibodies
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Verify HRP activity: Include positive controls to confirm enzyme functionality
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Consider cross-reactivity: Test for potential cross-reactivity with other SLC family members
Document all experimental conditions systematically to isolate variables causing inconsistency .
What are the common pitfalls in interpreting SLC1A7 localization data from immunofluorescence studies?
Common pitfalls in SLC1A7 localization studies include:
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Autofluorescence in tissues (particularly in retina): Implement appropriate controls and autofluorescence quenching
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Background from non-specific binding: Optimize blocking conditions and antibody dilutions
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Cross-reactivity with other glutamate transporters: Validate with peptide competition assays
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Fixation artifacts affecting membrane protein localization: Compare multiple fixation methods
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Over-interpretation of co-localization: Use appropriate statistical measures (Pearson's coefficient, Manders' overlap)
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Misattribution of subcellular localization: Confirm with subcellular fractionation or super-resolution microscopy
Always include proper controls and use complementary approaches to confirm localization findings .
How can I distinguish between specific and non-specific binding when using SLC1A7 HRP-conjugated antibodies?
To distinguish specific from non-specific binding:
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Peptide competition assay: Pre-incubate antibody with excess immunizing peptide (AA 115-220)
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Concentration gradient analysis: Perform titration experiments to identify optimal antibody concentration
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Negative control tissues: Use tissues known not to express SLC1A7
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Isotype control: Use matched IgG at the same concentration
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Multiple antibodies approach: Compare staining patterns using antibodies targeting different epitopes
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Knockout/knockdown validation: Compare staining in control versus SLC1A7-depleted samples
The absence of signal in peptide-blocked and knockout/knockdown samples provides strong evidence for specificity .
How can SLC1A7 HRP-conjugated antibodies be utilized in studying the role of glutamate transporters in T-cell biology?
Recent research has highlighted the importance of amino acid transporters in T-cell activation and differentiation. While SLC1A7 is not among the most studied transporters in this context (compared to LAT1, ASCT2, and GAT-1), HRP-conjugated SLC1A7 antibodies can be employed to:
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Investigate potential expression of SLC1A7 in different T-cell subsets
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Examine changes in SLC1A7 expression during T-cell activation
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Correlate SLC1A7 expression with mTORC1 signaling
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Compare expression patterns across different immune cell populations
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Study potential functional roles in glutamate sensing or transport in immune contexts
This represents an emerging research direction connecting neurotransmitter transport systems with immune function .
What considerations should be made when using SLC1A7 antibodies for studying "missing proteins"?
When investigating SLC1A7 in the context of missing proteins (proteins with limited or no experimental evidence):
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Implement enhanced validation approaches as described in the literature
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Use multiple antibodies targeting different epitopes
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Combine antibody-based methods with MS/MS approaches
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Focus on tissues with predicted expression based on transcriptomic data
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Consider using inducible expression systems to validate antibody specificity
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Document all validation steps meticulously
This approach increases confidence in protein identification and characterization, particularly for proteins with limited prior evidence .
