TMEM87B Antibody, FITC conjugated

What is TMEM87B and what cellular functions does it participate in?

TMEM87B (Transmembrane protein 87B) is a multi-pass membrane protein that plays significant roles in various cellular processes. Based on current research, TMEM87B is involved in cell proliferation, cell migration, and signal transduction pathways . The protein is expressed in multiple human tissues and cell types, making it a potentially important target for understanding fundamental cell biology.

The significance of TMEM87B extends to several research areas including cancer biology, developmental biology, and neurobiology, as its involvement in these essential cellular functions suggests potential roles in both physiological and pathological conditions . The molecular weight of the native TMEM87B protein is approximately 63-70 kDa, though this may vary slightly depending on post-translational modifications and experimental conditions .

Future investigations into TMEM87B function may contribute to the development of novel therapeutic strategies targeting various disease processes that involve altered cell proliferation and signaling pathways.

What is FITC conjugation and how does it benefit antibody applications?

FITC (Fluorescein isothiocyanate) conjugation is a chemical process that covalently attaches the fluorescent FITC molecule to antibodies, enabling visualization in various fluorescence-based applications. The conjugation occurs primarily through the reaction between the isothiocyanate group of FITC and primary amines (particularly lysine residues) on the antibody protein . This labeling allows researchers to track and visualize the antibody-antigen binding events using fluorescence microscopy, flow cytometry, and other fluorescence-based detection methods.

The benefits of FITC conjugation include:

• Direct detection without secondary antibodies, streamlining experimental protocols

• Strong fluorescence signal with excitation maximum at approximately 495 nm and emission at 519 nm

• Compatibility with standard FITC filter sets in most fluorescence imaging systems

• Well-established conjugation protocols with predictable fluorescein/protein (F/P) ratios

• Ability to perform multicolor experiments when combined with other fluorophores

Research indicates that optimal FITC conjugation can be achieved under specific conditions: reaction temperature at room temperature, pH 9.5, and protein concentration around 25 mg/ml, with maximal labeling typically occurring within 30-60 minutes . The quality of the conjugation significantly impacts experimental outcomes, making proper conjugation techniques essential for reliable results.

What applications is TMEM87B antibody, FITC conjugated suitable for?

TMEM87B antibody with FITC conjugation is suitable for multiple research applications that require fluorescent detection. Based on validated product information, this antibody has demonstrated utility in several key techniques:

• ELISA (Enzyme-Linked Immunosorbent Assay): The antibody can be used at dilutions ranging from 1:2000 to 1:10000 for ELISA applications, allowing quantitative detection of TMEM87B protein in various sample types .

• Immunofluorescence microscopy: The FITC conjugation enables direct visualization of TMEM87B in fixed cells and tissue sections, providing insights into subcellular localization and expression patterns.

• Flow cytometry: The antibody can be used to detect and quantify TMEM87B expression in cell populations, potentially at the single-cell level.

• Immunohistochemistry (IHC): Some TMEM87B antibodies have been validated for IHC applications at dilutions of 1:200 to 1:500, though specific validation for the FITC-conjugated version should be confirmed .

When designing experiments, researchers should consider the specific reactivity of the antibody, which has been validated for human samples as indicated in the product specifications . Cross-reactivity with other species should be verified through preliminary experiments before proceeding with full studies.

How should TMEM87B antibody, FITC conjugated be stored and handled?

Proper storage and handling of FITC-conjugated TMEM87B antibody is crucial for maintaining its activity and fluorescence properties. Based on manufacturer specifications, the following guidelines should be implemented:

• Storage temperature: Upon receipt, store the antibody at -20°C or -80°C for long-term stability . The lower temperature (-80°C) is preferable for extended storage periods.

• Aliquoting: To prevent repeated freeze-thaw cycles that can damage both the antibody and the fluorophore, divide the stock solution into small single-use aliquots before freezing .

• Buffer conditions: The antibody is typically provided in a stabilizing buffer containing 50% glycerol, 0.01M PBS at pH 7.4, and 0.03% Proclin 300 as a preservative . This formulation helps maintain antibody integrity during storage.

• Light protection: FITC is sensitive to photobleaching, so the antibody should be protected from light during all handling steps. Use amber tubes for storage and minimize exposure to light during experimental procedures.

• Freeze-thaw considerations: Avoid repeated freeze-thaw cycles as these can significantly reduce antibody activity and fluorescence intensity . Ideally, limit to no more than 3-5 cycles.

• Working solution stability: Once diluted for experiments, the antibody should be used within 24 hours for optimal performance. Prepare working dilutions immediately before use whenever possible.

Following these storage and handling recommendations will help ensure consistent and reliable results when using FITC-conjugated TMEM87B antibody in research applications.

What are the optimal conditions for FITC conjugation to antibodies targeting transmembrane proteins?

Optimizing FITC conjugation to antibodies targeting transmembrane proteins like TMEM87B requires careful control of multiple parameters to achieve ideal labeling efficiency while preserving antibody functionality. Research on FITC conjugation indicates several critical factors that influence conjugation success:

The reaction conditions should be precisely controlled with optimal parameters including: room temperature incubation, pH 9.5 buffer environment, and protein concentration of approximately 25 mg/ml . Under these conditions, maximal labeling is typically achieved within 30-60 minutes, striking an appropriate balance between sufficient conjugation and minimizing over-labeling that could compromise antibody function .

The molecular fluorescein/protein (F/P) ratio is a critical quality metric for conjugated antibodies. The ideal F/P ratio depends on the specific application:

• For immunofluorescence microscopy: 2-4 FITC molecules per antibody provides sufficient brightness while minimizing background

• For flow cytometry: 4-7 FITC molecules per antibody often yields optimal signal-to-noise ratios

• For ELISA applications: 1-3 FITC molecules per antibody typically provides adequate detection capability

Advanced purification methods, particularly gradient DEAE Sephadex chromatography, can effectively separate optimally labeled antibodies from under- and over-labeled proteins, ensuring a homogeneous preparation with consistent performance characteristics . This separation step is especially important for applications requiring precise quantification or high sensitivity.

When working specifically with antibodies against transmembrane proteins like TMEM87B, additional considerations include preserving epitope recognition in the transmembrane or extracellular domains, which may require modified conjugation approaches to prevent steric hindrance at the antigen-binding site.

How can researchers validate the specificity of TMEM87B antibody in experimental systems?

Validating the specificity of TMEM87B antibody, particularly when FITC-conjugated, requires a multi-faceted approach to ensure reliable and reproducible results. Comprehensive validation strategies include:

Western Blot Validation:
Western blot analysis serves as a primary validation method, where the antibody should detect a specific band at approximately 63-70 kDa corresponding to TMEM87B . Validation across multiple cell lines and tissue types helps confirm consistency of detection. For example, testing across human cell lines such as T-47D, MDA-MB-453, PC-3, and MCF-7, as well as in brain tissue lysates from different species, can demonstrate specificity and potential cross-reactivity .

Overexpression Systems:
Using TMEM87B overexpression lysates provides a powerful positive control. HEK293T cells transiently transfected with TMEM87B expression constructs (typically with tags such as C-Myc/DDK) can be used to validate antibody specificity by comparing signal intensity between transfected and non-transfected cells . This approach is particularly valuable for confirming that the antibody recognizes the target protein in its native conformation.

Immunohistochemistry (IHC) Cross-Validation:
IHC analysis in relevant tissues, such as human colon adenocarcinoma or liver cancer tissues (as used for the related TMEM87A), can provide spatial information about protein expression patterns . When performing IHC validation, heat-mediated antigen retrieval in EDTA buffer (pH 8.0) often provides optimal epitope exposure for transmembrane proteins.

Knockout/Knockdown Controls:
For definitive validation, CRISPR/Cas9-mediated knockout or siRNA-mediated knockdown of TMEM87B provides the most stringent control. The absence or significant reduction of signal in these negative control samples strongly supports antibody specificity.

Cross-Reactivity Assessment:
Testing potential cross-reactivity with closely related proteins, especially TMEM87A, is essential due to potential structural similarities. Parallel testing with both TMEM87A and TMEM87B specific antibodies on the same samples can reveal any cross-reactivity issues.

Fluorescence Colocalization:
For FITC-conjugated antibodies specifically, colocalization studies using differentially labeled antibodies against the same target or against proteins known to interact with TMEM87B can provide additional evidence of specificity in cellular contexts.

What are the critical considerations for designing flow cytometry experiments with FITC-conjugated TMEM87B antibody?

Designing flow cytometry experiments with FITC-conjugated TMEM87B antibody requires careful planning to ensure accurate detection and quantification. The following considerations are essential for robust experimental design:

Antibody Titration:
Begin by performing antibody titration to determine the optimal concentration that provides maximal specific signal with minimal background. Starting with the manufacturer's recommended dilutions (e.g., 1:2000-1:10000 for ELISA applications), test a range of concentrations in your specific cell types . Plot the signal-to-noise ratio against antibody concentration to identify the optimal working dilution.

Panel Design:
When incorporating FITC-conjugated TMEM87B antibody into multicolor panels, consider:

• FITC emission spectrum (peak ~519 nm) and potential spectral overlap with other fluorophores

• Appropriate compensation controls when using multiple fluorophores

• Expression level of TMEM87B relative to other target proteins (allocate brighter fluorophores to lower-expressed targets)

Cell Preparation Protocols:
For transmembrane proteins like TMEM87B, the cell preparation method significantly impacts detection:

• For surface epitopes: Use gentle fixation (0.5-2% paraformaldehyde) or stain live cells

• For intracellular/transmembrane domains: Use permeabilization reagents compatible with membrane proteins (e.g., saponin-based buffers rather than harsh detergents)

• Optimize fixation time and temperature to preserve epitope recognition while ensuring adequate permeabilization

Controls:
Implement comprehensive controls including:

• Unstained cells to establish autofluorescence baseline

• Isotype control (FITC-conjugated rabbit IgG) to assess non-specific binding

• Fluorescence-minus-one (FMO) controls to set accurate gating boundaries

• Positive control samples (e.g., cells known to express high levels of TMEM87B)

• Negative control samples (e.g., TMEM87B-knockout cells if available)

Instrument Settings:
Configure flow cytometer settings appropriately:

• Excite FITC using the 488 nm laser

• Collect emission through a 530/30 nm bandpass filter

• Set PMT voltages to position negative population in the first decade of the display

• Ensure adequate dynamic range to discriminate between negative and positive populations

Sample Storage Considerations:
If analysis cannot be performed immediately after staining:

• Keep samples at 4°C and protected from light

• Analyze within 4-6 hours of staining when possible

• For longer storage, add additional fixative (0.1-0.5% paraformaldehyde) to preserve signal

How can researchers troubleshoot weak or non-specific signals when using FITC-conjugated TMEM87B antibodies?

Troubleshooting weak or non-specific signals with FITC-conjugated TMEM87B antibodies requires systematic evaluation of multiple experimental parameters. The following approaches address common issues:

Addressing Weak Signal Issues:

• Antibody Concentration and Incubation Conditions:

  • Increase antibody concentration incrementally, testing higher concentrations than the recommended range (e.g., up to 5-10 μg/ml for IHC applications)

  • Extend incubation time (e.g., from overnight at 4°C to 24-48 hours)

  • Optimize incubation temperature (4°C for longer incubations, room temperature or 37°C for shorter periods)

• Antigen Retrieval Enhancement:

  • For tissue sections or fixed cells, test different antigen retrieval methods:

    • Heat-mediated retrieval in EDTA buffer (pH 8.0) as used for related transmembrane proteins

    • Citrate buffer (pH 6.0) alternatives

    • Enzymatic retrieval with proteinase K (use with caution as over-digestion can destroy epitopes)

  • Increase retrieval time or temperature incrementally

• Signal Amplification Strategies:

  • Implement tyramide signal amplification (TSA) for significant signal enhancement

  • Use anti-FITC antibodies conjugated to brighter fluorophores or enzymes as a secondary detection step

  • Consider biotin-streptavidin amplification systems

• Fluorescence Preservation:

  • Add anti-fade reagents to mounting media

  • Use higher glycerol concentration (50%) in storage buffer to stabilize the antibody

  • Minimize exposure to light during all experimental steps

Resolving Non-specific Signal Problems:

• Blocking Optimization:

  • Test increased blocking reagent concentration (e.g., 10% normal goat serum as used in validated protocols)

  • Extend blocking time from 30 minutes to 1-2 hours

  • Try alternative blocking agents (BSA, casein, commercial blocking buffers)

  • Include 0.1-0.3% Triton X-100 in blocking buffer to reduce non-specific membrane binding

• Washing Protocol Enhancement:

  • Increase number of wash steps (5-6 washes instead of standard 3)

  • Extend wash duration (10-15 minutes per wash)

  • Add detergent (0.05-0.1% Tween-20) to wash buffers

  • Use TBS-0.1% Tween for washing as in validated Western blot protocols

• Antibody Validation:

  • Test antibody specificity using positive control lysates (e.g., TMEM87B overexpression lysates)

  • Perform absorption controls by pre-incubating antibody with recombinant TMEM87B protein

  • Compare staining pattern with alternative antibodies against the same target

  • Verify antibody specificity using knockout/knockdown approaches

• Sample-specific Considerations:

  • Reduce autofluorescence using treatments like Sudan Black B (0.1-0.3%)

  • For tissues with high endogenous biotin, use avidin/biotin blocking kits

  • Quench endogenous peroxidase activity with hydrogen peroxide treatment if using HRP-based detection systems

What are the quantitative parameters for assessing FITC conjugation quality to TMEM87B antibodies?

The quality assessment of FITC conjugation to TMEM87B antibodies involves evaluating several quantitative parameters that directly impact experimental performance. Researchers should consider the following metrics when assessing conjugation quality:

Fluorescein/Protein (F/P) Ratio:
The F/P ratio is the most critical parameter for assessing conjugation efficiency. Optimal ratios typically range between 2-7 FITC molecules per antibody molecule, depending on the application . This ratio can be calculated using spectrophotometric measurements:

$\text{F/P ratio} = \frac{A_{495} \times MW_{IgG}}{195,000 \times A_{280} - (0.35 \times A_{495})}$

Where:

• A₄₉₅ is the absorbance at 495 nm (FITC peak)

• A₂₈₀ is the absorbance at 280 nm (protein peak)

• MW_IgG is the molecular weight of IgG (approximately 150,000 Da)

• 195,000 is the molar extinction coefficient of FITC

• 0.35 is the correction factor for FITC contribution at 280 nm

Protein Recovery:
Quantify the percentage of protein recovered after conjugation and purification compared to the starting material. Efficient conjugation protocols should yield recovery rates of >80% . Lower recovery rates may indicate protein loss, aggregation, or precipitation during the conjugation process.

Absorbance Spectrum Profile:
Record full absorbance spectra (250-550 nm) to assess:

• Peak ratio (A₄₉₅/A₂₈₀): Values between 0.3-0.7 typically indicate good conjugation

• Spectrum shape: Should show distinct peaks at 280 nm (protein) and 495 nm (FITC)

• Absence of shoulders or abnormal peaks that might indicate protein aggregation or contaminants

Fluorescence Quantum Yield:
Measure fluorescence intensity relative to standard FITC solutions of known concentration. Higher quantum yields indicate better conjugation with minimal quenching. This can be calculated using:

$\Phi_{sample} = \Phi_{standard} \times \frac{I_{sample}}{I_{standard}} \times \frac{A_{standard}}{A_{sample}}$

Where:

• Φ represents quantum yield

• I represents integrated fluorescence intensity

• A represents absorbance at the excitation wavelength

Degree of Labeling Homogeneity:
Use analytical techniques such as size-exclusion HPLC or capillary electrophoresis to assess the distribution of F/P ratios in the antibody preparation. A narrow distribution indicates uniform conjugation, while broad or multi-modal distributions suggest heterogeneous labeling that may impact experimental consistency.

Antibody Functionality Tests:
Quantitatively measure:

• Antigen binding capacity (through ELISA) compared to unconjugated antibody

• Specifically for TMEM87B antibodies, compare ELISA reactivity between conjugated and unconjugated versions at equivalent concentrations

• Signal-to-noise ratio in flow cytometry or immunofluorescence applications

Storage Stability Assessment:
Measure fluorescence retention over time under recommended storage conditions (-20°C or -80°C) . Calculate the percentage of fluorescence retention after defined time intervals (1 week, 1 month, 3 months, 6 months) to establish shelf-life expectations. Properly conjugated antibodies should retain >80% of initial fluorescence after 6 months at -80°C.

What protocol should be followed for immunofluorescence staining using FITC-conjugated TMEM87B antibody?

The following detailed protocol provides step-by-step guidance for immunofluorescence staining using FITC-conjugated TMEM87B antibody, incorporating validated parameters from research on similar transmembrane proteins:

Materials Required:

• FITC-conjugated TMEM87B antibody

• PBS (phosphate-buffered saline), pH 7.4

• Fixation solution: 4% paraformaldehyde in PBS

• Permeabilization solution: 0.1-0.3% Triton X-100 in PBS

• Blocking solution: 10% normal goat serum in PBS

• Antigen retrieval buffer: EDTA buffer (pH 8.0) or citrate buffer (pH 6.0)

• DAPI or other nuclear counterstain

• Anti-fade mounting medium

• Microscope slides and coverslips

Protocol:

• Sample Preparation:

  • For cultured cells: Seed cells on sterile coverslips or chamber slides 24-48 hours before staining

  • For tissue sections: Prepare 5-7 μm paraffin or frozen sections on positively charged slides

• Fixation:

  • Cultured cells: Remove media, wash with PBS, and fix with 4% paraformaldehyde for 15 minutes at room temperature

  • Tissue sections: Deparaffinize (if paraffin-embedded) following standard protocols before proceeding to antigen retrieval

• Antigen Retrieval (for tissue sections and fixed cells if needed):

  • Heat-mediated retrieval in EDTA buffer (pH 8.0) at 95-98°C for 15-20 minutes

  • Allow to cool at room temperature for 20 minutes

• Permeabilization:

  • Incubate samples with 0.1-0.3% Triton X-100 in PBS for 10 minutes at room temperature

  • Wash 3 times with PBS, 5 minutes each

• Blocking:

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

  • For tissues with high background, extend blocking to 2 hours or include 0.1% Tween-20 in the blocking solution

• Primary Antibody Incubation:

  • Dilute FITC-conjugated TMEM87B antibody in blocking solution at 1:200-1:500 (for IHC-validated antibodies)

  • For ELISA-validated antibodies, start with higher concentrations (1:100) and optimize

  • Incubate overnight at 4°C in a humidified chamber protected from light

  • For weak signals, extend incubation to 24-48 hours at 4°C

• Washing:

  • Wash 5 times with PBS containing 0.1% Tween-20, 5 minutes each

  • Perform all washing steps protected from light to prevent photobleaching

• Nuclear Counterstaining:

  • Incubate with DAPI (1 μg/ml in PBS) for 5 minutes at room temperature

  • Wash 3 times with PBS, 5 minutes each

• Mounting:

  • Mount coverslips on slides (or add coverslips to slides) using anti-fade mounting medium

  • Seal edges with clear nail polish to prevent drying

  • Allow mounting medium to set for at least 2 hours before imaging

• Imaging and Analysis:

  • Image using epifluorescence or confocal microscopy with appropriate filter sets:

    • FITC: excitation ~490 nm, emission ~525 nm

    • DAPI: excitation ~350 nm, emission ~460 nm

  • Capture images using consistent exposure times for quantitative comparisons

  • Use appropriate software for colocalization or intensity analysis

Controls and Validation:

• Include isotype control (FITC-conjugated rabbit IgG) at the same concentration

• Include positive control samples (tissues/cells known to express TMEM87B)

• For validation, compare staining pattern with other TMEM87B antibodies or with known TMEM87B-interacting proteins

How does FITC conjugation affect the binding kinetics and affinity of TMEM87B antibodies?

FITC conjugation can significantly impact the binding kinetics and affinity of TMEM87B antibodies through several molecular mechanisms. Understanding these effects is crucial for accurate experimental design and interpretation of results:

Impact on Antibody-Antigen Binding Kinetics:

FITC conjugation typically modifies lysine residues on the antibody, which can alter both the association (k_on) and dissociation (k_off) rates that govern antibody-antigen interactions. Research on antibody conjugation indicates that:

• Association rate (k_on) may decrease by 10-30% compared to unconjugated antibodies due to:

  • Steric hindrance from the FITC molecules

  • Altered electrostatic interactions (FITC introduces negative charges)

  • Conformational changes in the antibody structure

• Dissociation rate (k_off) is often more significantly affected, potentially increasing by 20-50%, leading to:

  • Reduced residence time of the antibody on its target

  • Potential loss of apparent affinity in dynamic systems

  • Greater sensitivity to stringent washing conditions

Affinity Changes and Molecular Basis:

• At low F/P ratios (1-3): Minimal affinity reduction, typically 0-20%

• At moderate F/P ratios (4-7): Moderate affinity reduction, typically 20-40%

• At high F/P ratios (>8): Substantial affinity reduction, often >50%

The molecular basis for these changes involves:

• Modification of lysine residues in or near antigen-binding regions

• Altered protein folding dynamics due to the hydrophobic nature of the FITC molecule

• Changes in the antibody's isoelectric point affecting electrostatic interactions

Experimental Considerations for TMEM87B Detection:

For TMEM87B antibodies specifically, researchers should consider:

• Epitope accessibility: If the antibody targets regions of TMEM87B that are partially embedded in the membrane, FITC conjugation may disproportionately affect binding due to additional steric constraints.

• Buffer optimization: Studies have indicated that maintaining proper pH (7.4) and ionic strength in experimental buffers is critical for preserving the binding functionality of FITC-conjugated antibodies .

• Incubation time adjustments: To compensate for potentially reduced affinity, researchers may need to:

  • Extend primary antibody incubation times (from standard overnight to 24-48 hours)

  • Optimize antibody concentration (typically requiring 1.5-2× higher concentrations than unconjugated versions)

  • Reduce washing stringency (shorter wash times or reduced detergent concentration)

Quantitative Assessment Methods:

Researchers can quantitatively assess the impact of FITC conjugation on their specific TMEM87B antibody using:

• Surface Plasmon Resonance (SPR) to measure:

  • K_d values before and after conjugation

  • Association and dissociation rate constants

• Competitive binding assays to determine:

  • IC₅₀ values for conjugated versus unconjugated antibodies

  • Relative affinity measurements under identical conditions

• Saturation binding experiments to establish:

  • Bmax (maximum binding capacity)

  • Apparent K_d under experimental conditions

By understanding these principles and conducting appropriate validation experiments, researchers can account for the effects of FITC conjugation on TMEM87B antibody performance and design experiments accordingly.

What strategies can optimize ELISA sensitivity when using FITC-conjugated TMEM87B antibodies?

Optimizing ELISA sensitivity with FITC-conjugated TMEM87B antibodies requires careful consideration of both the fluorescence properties of FITC and the specific characteristics of TMEM87B detection. The following strategies can significantly enhance sensitivity, specificity, and reproducibility:

Assay Format Optimization:

• Sandwich ELISA Configuration:

  • Use a capture antibody specific to a different epitope of TMEM87B

  • Apply the FITC-conjugated TMEM87B antibody as the detection antibody

  • This approach typically provides higher specificity and sensitivity than direct ELISA formats

• Signal Amplification Systems:

  • Implement anti-FITC antibodies conjugated to HRP or other enzymes

  • Use tyramide signal amplification (TSA) for 10-100× signal enhancement

  • Consider biotin-streptavidin systems with multiple enzyme molecules per binding event

• Microplate Selection:

  • Use high-binding, black microplates for fluorescence-based detection

  • White microplates for chemiluminescence if converting fluorescence to luminescence detection

  • Consider specialized plates with enhanced protein binding characteristics

Technical Parameter Optimization:

• Antibody Concentration Titration:

  • Perform checkerboard titrations to identify optimal antibody concentration

  • Start with the manufacturer's recommended dilution range (1:2000-1:10000)

  • Test at least 5-7 different dilutions to identify the optimal signal-to-noise ratio

• Buffer Composition:

  • Optimize blocking buffer (5-10% BSA or normal goat serum)

  • Add 0.05% Tween-20 to reduce non-specific binding

  • Include stabilizers such as 5-10% glycerol in dilution buffers

• Incubation Parameters:

  • Extend primary antibody incubation time (overnight at 4°C)

  • Optimize temperature (4°C for higher specificity, room temperature for faster kinetics)

  • Use orbital shaking (50-100 rpm) during incubations to enhance binding

• Washing Protocol Refinement:

  • Increase wash cycles (5-6 times rather than standard 3)

  • Use TBS-0.1% Tween for washing as in validated protocols

  • Implement automated washers for consistent washing pressure and timing

Signal Detection Optimization:

• Fluorescence-based Detection:

  • Use excitation at 485-495 nm and emission at 515-525 nm for optimal FITC detection

  • Adjust gain settings on fluorescence plate readers for maximum sensitivity

  • Include standard curves with known amounts of FITC-conjugated antibody for quantification

• Enzyme-based Detection Alternatives:

  • Convert to HRP-based detection using anti-FITC-HRP secondary antibodies

  • Select optimal substrates (TMB for colorimetric, enhanced chemiluminescence for luminescence)

  • Extend substrate development time for low-abundance targets

• Data Analysis Approaches:

  • Use 4-parameter logistic curve fitting for standard curves

  • Implement background subtraction using blank wells

  • Calculate coefficient of variation (CV) values for replicates (<15% for acceptable precision)

TMEM87B-Specific Considerations:

• Antigen Capture Strategy:

  • For transmembrane proteins like TMEM87B, use detergent-solubilized preparations

  • Consider using recombinant fragments corresponding to extracellular domains

  • Test both native and denatured protein forms to determine optimal detection conditions

• Sample Preparation:

  • For cell lysates, use modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA) with protease inhibitors

  • Centrifuge lysates at high speed (>10,000×g) to remove insoluble material

  • Normalize total protein concentration across samples (BCA assay recommended)

• Validation Controls:

  • Include TMEM87B overexpression lysates as positive controls

  • Use parental cell line lysates as negative or baseline controls

  • Create standard curves using recombinant TMEM87B protein when available

By implementing these optimization strategies, researchers can achieve significantly improved sensitivity and specificity in ELISA assays using FITC-conjugated TMEM87B antibodies, potentially reaching detection limits in the low picogram range.