TMEM82 Antibody

What is TMEM82 and where is it primarily expressed?

TMEM82 (Transmembrane Protein 82) is a protein encoded by the TMEM82 gene located on chromosome 1 (1p36.21). The gene spans 5,484 base pairs, contains 6 exons and 5 introns, and encodes a 343 amino acid protein with a molecular weight of approximately 37 kDa. The protein features 8 predicted transmembrane domains and 1 disordered region. Expression analysis shows high levels in kidney, liver, small intestine, and duodenum, with moderate expression in colon and stomach. During fetal development, TMEM82 is expressed in adrenal and heart tissues, with significant upregulation in the small intestine by 15 weeks of gestation .

What types of TMEM82 antibodies are available for research?

Several types of TMEM82 antibodies are available for research purposes, targeting different regions of the protein. These include antibodies recognizing amino acids 168-202, amino acids 72-121, and the N-terminal region. Most are polyclonal antibodies raised in rabbits, though some mouse-derived polyclonal options exist. These antibodies are available in various formats: unconjugated, HRP-conjugated, FITC-conjugated, and biotin-conjugated. Reactivity profiles vary by antibody but generally include human samples, with some cross-reacting with mouse, dog, rat, bat, pig, and rabbit samples .

What experimental applications are TMEM82 antibodies suitable for?

TMEM82 antibodies can be utilized in multiple experimental applications depending on their specific formulation. Common applications include enzyme-linked immunosorbent assay (ELISA), western blotting (WB), immunohistochemistry (IHC), and immunofluorescence (IF). The recommended dilutions vary by application; for instance, ELISA typically requires a 1:62500 dilution, while western blotting generally calls for 1 μg/mL concentration with HRP-conjugated secondary antibodies at 1:50,000-100,000 dilution .

What controls should be included when using TMEM82 antibodies in experimental procedures?

For robust experimental design with TMEM82 antibodies, include these essential controls:

• Positive tissue controls: Include samples from kidney, liver, small intestine, or duodenum where TMEM82 is highly expressed

• Negative controls: Include tissues with minimal TMEM82 expression or use blocking peptides

• Antibody controls: Include a primary antibody isotype control and a secondary antibody-only control

• Knockdown/knockout validation: Where possible, include samples from TMEM82 knockdown/knockout models

• Recombinant protein: Use purified recombinant TMEM82 protein as a positive control for western blotting

These controls help validate antibody specificity and experimental validity.

How can I effectively design experiments to study TMEM82's role during embryonic development?

To investigate TMEM82's role in embryonic development, design a temporal and spatial expression study. Based on known fetal expression patterns, focus on adrenal, heart, and small intestinal tissues at various developmental stages, with particular attention to the 15-week gestational period when small intestinal expression increases significantly . Employ both protein detection methods (western blot, IHC) and transcript analysis (qPCR, in situ hybridization). For functional studies, consider siRNA knockdown in embryonic cell lines or organoid cultures derived from relevant tissues. Time-course experiments tracking TMEM82 expression alongside developmental markers will be particularly informative. For animal models, carefully select antibodies with appropriate species reactivity across human, mouse, or rat samples to ensure experimental consistency .

What is the optimal protocol for using TMEM82 antibodies in western blotting?

For optimal western blotting with TMEM82 antibodies, follow this methodological approach:

• Sample preparation: Extract proteins using RIPA buffer supplemented with protease inhibitors; sonicate briefly to disrupt transmembrane proteins

• Protein separation: Use 10-12% SDS-PAGE gels; load 20-30 μg protein per lane

• Transfer: Employ semi-dry transfer to PVDF membrane (recommended over nitrocellulose for transmembrane proteins)

• Blocking: Block with 5% non-fat milk in TBST for 1 hour at room temperature

• Primary antibody: Dilute TMEM82 antibody to 1 μg/mL in blocking solution; incubate overnight at 4°C

• Washing: Wash membrane 3×10 minutes with TBST

• Secondary antibody: Apply HRP-conjugated secondary antibody at 1:50,000-100,000 dilution; incubate for 1 hour at room temperature

• Detection: Use enhanced chemiluminescence; expect a band at approximately 37 kDa

• Controls: Include kidney or liver lysate as positive control

For troubleshooting membrane proteins, consider using specialized membrane protein extraction buffers or gentler detergents if signal is weak.

How should TMEM82 antibodies be used for immunohistochemistry on tissue sections?

For effective immunohistochemistry with TMEM82 antibodies, follow this methodological approach:

• Fixation: Fix tissues in 10% neutral buffered formalin for 24 hours

• Processing: Embed in paraffin and section at 4-5 μm thickness

• Deparaffinization: Use standard xylene and graded alcohol series

• Antigen retrieval: Perform heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes

• Endogenous peroxidase blocking: Treat with 3% hydrogen peroxide for 10 minutes

• Protein blocking: Use 5% normal serum (matching secondary antibody species) for 1 hour

• Primary antibody: Apply TMEM82 antibody at manufacturer-recommended dilution (typically 1:100-1:500); incubate overnight at 4°C

• Secondary antibody: Use appropriate HRP-conjugated secondary antibody; incubate for 1 hour at room temperature

• Visualization: Develop with DAB substrate and counterstain with hematoxylin

• Analysis: Focus on expected subcellular localization (membrane-associated pattern)

Optimize antibody dilution and antigen retrieval method for each specific TMEM82 antibody clone and tissue type.

What is the recommended procedure for ELISA using TMEM82 antibodies?

For optimal ELISA using TMEM82 antibodies, follow this protocol:

• Plate coating: Coat 96-well plates with capture antibody (1-2 μg/mL in carbonate buffer, pH 9.6); incubate overnight at 4°C

• Blocking: Block with 1-2% BSA in PBS for 1-2 hours at room temperature

• Samples and standards: Add recombinant TMEM82 protein standards and samples; incubate for 2 hours at room temperature

• Detection antibody: Add TMEM82 antibody diluted at 1:62500; incubate for 2 hours at room temperature

• Secondary antibody: Add appropriate HRP-conjugated secondary antibody; incubate for 1 hour at room temperature

• Substrate: Add TMB substrate and monitor color development

• Stop reaction: Add stop solution (2N H₂SO₄) when appropriate color intensity is reached

• Analysis: Read absorbance at 450 nm and calculate TMEM82 concentration using standard curve

For sandwich ELISA, ensure the capture and detection antibodies recognize different epitopes of TMEM82 to prevent competitive binding.

How should I interpret western blot results when using TMEM82 antibodies?

When interpreting western blot results with TMEM82 antibodies, consider these analytical guidelines:

• Expected molecular weight: The primary band should appear at approximately 37 kDa, corresponding to the predicted molecular weight of TMEM82

• Multiple bands: As a multi-transmembrane protein, TMEM82 may show additional bands representing glycosylated forms or proteolytic fragments; validate these using appropriate controls

• Tissue-specific patterns: Expect stronger signals in kidney, liver, small intestine, and duodenum samples compared to other tissues

• Quantitative analysis: When performing densitometry, normalize TMEM82 signal to appropriate housekeeping proteins (β-actin for cytosolic fraction, Na⁺/K⁺-ATPase for membrane fraction)

• Sample preparation artifacts: Membrane proteins can aggregate during boiling; if smearing occurs at high molecular weights, reduce heating time or temperature during sample preparation

Compare your results with available literature data on TMEM82 expression patterns to validate your findings.

What are the common challenges in interpreting immunohistochemistry results for TMEM82?

Researchers frequently encounter these challenges when interpreting TMEM82 immunohistochemistry results:

• Subcellular localization: TMEM82, with its 8 transmembrane domains, should display a membrane-associated pattern; cytoplasmic staining may indicate antibody cross-reactivity or protein trafficking

• Background staining: Distinguish non-specific background from true signal by comparing with negative controls and isotype controls

• Cell-type specificity: Validate cell-type specific expression patterns (e.g., in parietal cells, proximal tubular cells, hepatocytes) using co-staining with established cell-type markers

• Signal intensity variation: Account for fixation artifacts, specimen quality, and antigen retrieval effectiveness when interpreting intensity differences

• Epitope masking: Some epitopes may be inaccessible in certain tissue preparations; compare results using antibodies targeting different TMEM82 regions (N-terminal vs. AA 168-202)

Document both positive and negative staining patterns across different cell types within the same tissue section to provide internal validation.

How can I determine the specificity of my TMEM82 antibody results?

To determine the specificity of TMEM82 antibody results, implement these analytical approaches:

• Multi-antibody validation: Compare results from antibodies targeting different epitopes of TMEM82 (e.g., N-terminal vs. mid-region)

• Peptide competition: Pre-incubate the antibody with the immunizing peptide; specific signals should be abolished

• Genetic validation: Compare results between wildtype and TMEM82 knockout/knockdown samples

• Correlation with mRNA: Compare protein expression patterns with TMEM82 mRNA expression data from public databases or your own qPCR results

• Recombinant protein control: Include purified recombinant TMEM82 protein as a positive control in western blots

• Cross-species consistency: Evaluate whether the expression pattern is conserved across species in a manner consistent with evolutionary conservation

How can structural mapping approaches enhance TMEM82 antibody research?

Structural mapping approaches can significantly enhance TMEM82 antibody research by:

• Epitope prediction: Using the Structurally Annotating Antibodies (SAAB) pipeline to predict three-dimensional epitope configurations on TMEM82, enabling more rational antibody selection

• Paratope characterization: Mapping the complementarity-determining regions (CDRs) of TMEM82 antibodies to understand the physicochemical basis of binding specificity

• Binding affinity prediction: Correlating sequence identity with structural similarity to estimate binding properties between antibodies and TMEM82 variants

• Cross-reactivity analysis: Predicting potential cross-reactivity with related proteins based on structural homology

• Immunogenicity assessment: Identifying potentially immunogenic regions of TMEM82 for antibody development by analyzing the exposed epitopes in the predicted 3D structure

Implementing these approaches requires integrating sequence data with structural biology tools, as detailed in frameworks like SAAB that have been successfully applied to antibody repertoire analysis .

What approaches can be used to study TMEM82 protein-protein interactions using antibodies?

To investigate TMEM82 protein-protein interactions using antibodies, consider these advanced methodological approaches:

• Co-immunoprecipitation (Co-IP): Use TMEM82 antibodies for pull-down experiments followed by mass spectrometry to identify interaction partners

• Proximity labeling: Couple TMEM82 antibodies with enzymes like BioID or APEX2 to biotinylate proximal proteins for subsequent identification

• PLA (Proximity Ligation Assay): Utilize TMEM82 antibodies alongside antibodies against suspected interaction partners to visualize interactions in situ with single-molecule resolution

• FRET/BRET analysis: Label TMEM82 antibodies with donor fluorophores and potential interaction partners with acceptor fluorophores to monitor energy transfer

• Crosslinking immunoprecipitation: Stabilize transient interactions using chemical crosslinkers before immunoprecipitation with TMEM82 antibodies

For all these approaches, carefully validate antibody specificity using the controls described earlier, and consider the membrane-embedded nature of TMEM82 when designing extraction and interaction conditions .

How can TMEM82 antibodies be used in studying pathological conditions?

TMEM82 antibodies can be leveraged to investigate pathological conditions through these advanced research approaches:

• Expression profiling: Compare TMEM82 levels between normal and diseased tissues (particularly in kidney, liver, and gastrointestinal disorders) using immunohistochemistry and western blotting

• Biomarker development: Evaluate TMEM82 as a potential diagnostic marker in conditions affecting tissues with high TMEM82 expression; develop sandwich ELISA methods for serum or tissue lysate analysis

• Genetic variant correlation: Investigate how known TMEM82 genetic variants (such as the SNP associated with Insulin-Like Growth Factor Binding Protein 3) affect protein expression and localization using variant-specific antibodies

• Therapeutic target validation: Use TMEM82 antibodies to evaluate the effectiveness of targeting TMEM82 in relevant disease models

• Signaling pathway analysis: Examine TMEM82's role in signaling pathways by analyzing post-translational modifications using modification-specific antibodies

Incorporate multi-omics approaches by correlating antibody-based findings with genomic, transcriptomic, and proteomic data to provide comprehensive mechanistic insights into TMEM82's role in disease processes.

What are common issues with western blotting for TMEM82 and how can they be resolved?

Common western blotting issues with TMEM82 antibodies and their solutions include:

Always verify results using positive control tissues known to express TMEM82, such as kidney or liver samples .

How can I troubleshoot immunohistochemistry issues when using TMEM82 antibodies?

For immunohistochemistry troubleshooting with TMEM82 antibodies, refer to this diagnostic table:

Start optimization with tissues known to have high TMEM82 expression (kidney, liver, small intestine) and consider examining cell types with known high expression such as proximal tubular cells or hepatocytes .

What strategies can overcome issues with antibody cross-reactivity in TMEM82 research?

To address antibody cross-reactivity issues in TMEM82 research, implement these methodological strategies:

• Multi-epitope validation: Compare results using antibodies targeting different regions of TMEM82 (N-terminal, AA 72-121, AA 168-202)

• Genetic knockout controls: Include samples from TMEM82 knockout models where available; any remaining signal indicates cross-reactivity

• Pre-absorption testing: Pre-incubate antibody with recombinant TMEM82 protein and related proteins to assess specific binding inhibition

• Western blot profiling: Analyze antibody reactivity across multiple tissues with varying TMEM82 expression levels to identify inconsistent signals

• Immunoprecipitation-mass spectrometry: Identify all proteins pulled down by the antibody to assess off-target binding

• Epitope mapping: Use peptide arrays to precisely define antibody binding sites and predict potential cross-reactivity

• Bioinformatic analysis: Compare the antibody's target sequence against the proteome to identify regions with high sequence similarity

For maximum confidence, combine multiple antibody validation approaches and document all validation steps meticulously in research publications.