SLC12A7 Antibody, FITC conjugated

What is SLC12A7 and why is it significant in research?

SLC12A7, also known as KCC4 (potassium-chloride cotransporter 4), is a member of the solute carrier family 12 that functions as an electroneutral potassium-chloride cotransporter. This transmembrane protein plays crucial roles in:

• Cell volume homeostasis

• Inorganic ion homeostasis

• Ion transmembrane transport

• Protein kinase binding activity

SLC12A7 is part of protein-containing complexes and contributes to several physiological processes. Its study is important for understanding fundamental cellular functions and potential implications in disease states where ion transport mechanisms may be dysregulated .

What are the primary research applications for SLC12A7 Antibody, FITC conjugated?

The FITC-conjugated SLC12A7 antibody serves multiple research applications:

• Flow cytometry: For quantitative analysis of SLC12A7 expression at the single-cell level

• Fluorescence microscopy: For visualization of SLC12A7 localization within cells and tissues

• Immunohistochemistry (IHC): For examining expression patterns in tissue sections

• Fluorescence-linked immunosorbent assay (FLISA): For protein quantification in solution

The FITC conjugation provides direct detection capability without requiring secondary antibodies, streamlining experimental workflows and reducing potential sources of background .

What antibody characteristics should researchers consider when selecting a SLC12A7 antibody?

When selecting a SLC12A7 antibody for research, consider these critical characteristics:

• Host species: Typically rabbit for SLC12A7 antibodies, which affects compatibility with other antibodies in multiplex experiments

• Isotype: Usually IgG, which influences binding properties and potential cross-reactivity

• Immunogen: The specific region of SLC12A7 used to generate the antibody (e.g., N-terminal region, Met1-Ser1083)

• Validation data: Flow cytometry validation comparing transfected versus non-transfected cells demonstrates specificity

• Conjugation: FITC provides green fluorescence (excitation ~495nm, emission ~519nm), suitable for standard fluorescence detection systems

• Purification method: Often purified by Protein A and peptide affinity chromatography to ensure specificity

How does FITC conjugation affect antibody functionality compared to unconjugated versions?

FITC conjugation introduces several important considerations for researchers:

• Direct detection: Eliminates need for secondary antibodies, reducing experiment time and potential background

• Spectral properties: Emits green fluorescence compatible with standard FITC filter sets (excitation ~495nm, emission ~519nm)

• Photostability: FITC is more susceptible to photobleaching than newer fluorophores like Alexa Fluors

• pH sensitivity: Fluorescence intensity can be affected by environmental pH, requiring consistent buffer conditions

• Antibody:fluorophore ratio: Degree of labeling affects brightness and potentially binding efficiency

• Storage requirements: Requires protection from light to maintain fluorescence intensity

• Application limitations: May be less suitable for applications requiring high photostability for extended imaging sessions

What flow cytometry protocols are recommended for optimal results with SLC12A7 Antibody, FITC conjugated?

For optimal flow cytometry results with FITC-conjugated SLC12A7 antibody:

Sample Preparation Protocol:

• Harvest cells (approximately 2×10⁶) by gentle dissociation

• Centrifuge at 300×g for 5 minutes

• Wash twice with ice-cold flow cytometry buffer (PBS + 2% FBS + 0.1% sodium azide)

• For surface staining only: Resuspend cells in 100μL flow cytometry buffer

• For intracellular staining: Fix and permeabilize cells according to manufacturer's protocol

Staining Protocol:

• Add optimized concentration of FITC-conjugated SLC12A7 antibody (typically 5-10μg/mL)

• Incubate for 30 minutes in the dark on ice

• Wash twice with ice-cold flow cytometry buffer

• Resuspend in 300μL flow cytometry buffer for analysis

• Analyze using flow cytometer with 488nm laser and appropriate FITC detection filter (typically 525/20nm)

Essential Controls:

• Unstained cells (for autofluorescence assessment)

• FITC-conjugated isotype control (for non-specific binding)

• Positive control (cells known to express SLC12A7 or transfected cells)

• FMO (Fluorescence Minus One) controls for multicolor panels

How can researchers verify SLC12A7 antibody specificity for confident data interpretation?

Rigorous validation of SLC12A7 antibody specificity is crucial for reliable research outcomes:

Recommended Validation Approaches:

• Genetic validation using transfected cells:

  • Compare staining in cells transfected with human SLC12A7 versus irrelevant transfectants

  • Example from similar antibody validation shows clear discrimination between positive and negative populations

• siRNA/shRNA knockdown validation:

  • Compare expression in cells with and without SLC12A7 knockdown

  • Quantify reduction in signal intensity corresponding to knockdown efficiency

• Western blot correlation (using corresponding non-conjugated antibody):

  • Confirm antibody recognizes protein of expected molecular weight

  • Compare to molecular weight markers and published literature

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Should observe dose-dependent reduction in specific staining

• Tissue expression patterns:

  • Confirm staining patterns match known SLC12A7 tissue distribution

  • Compare with mRNA expression data from public databases

What are the optimal immunohistochemistry protocols for SLC12A7 Antibody, FITC conjugated?

For successful immunohistochemistry with FITC-conjugated SLC12A7 antibody:

FFPE Tissue Protocol:

• Deparaffinization and rehydration:

  • Xylene: 2 × 10 minutes

  • 100% ethanol: 2 × 5 minutes

  • 95%, 80%, 70% ethanol: 3 minutes each

  • Distilled water: 5 minutes

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Pressure cooker method: 125°C for 30-45 seconds, then 90°C for 10 minutes

  • Allow to cool to room temperature (approximately 20 minutes)

• Permeabilization (if targeting intracellular domain):

  • 0.1-0.3% Triton X-100 in PBS for 10 minutes

  • Wash 3 × 5 minutes with PBS

• Blocking:

  • 10% normal serum (from species other than antibody host) + 1% BSA in PBS

  • Incubate 1 hour at room temperature in humid chamber

• Primary antibody incubation:

  • Apply FITC-conjugated SLC12A7 antibody (optimized concentration, typically 1-10 μg/mL)

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

  • Wash 3 × 5 minutes with PBS

• Counterstaining:

  • Apply DAPI nuclear counterstain (1 μg/mL) for 5 minutes

  • Wash 3 × 5 minutes with PBS

• Mounting:

  • Mount with anti-fade mounting medium specifically formulated for fluorescence

  • Seal edges of coverslip with nail polish or commercial sealant

  • Store slides at 4°C protected from light

Important optimization considerations:

• Titrate antibody to determine optimal concentration

• Test multiple antigen retrieval methods

• Include positive and negative control tissues

• Consider autofluorescence quenching steps for tissues with high natural fluorescence

How can researchers design effective multiplex experiments incorporating SLC12A7 Antibody, FITC conjugated?

Designing successful multiplex experiments requires strategic planning:

Fluorophore Selection Strategy:

Protocol Optimization Steps:

• Panel design:

  • Assign fluorophores based on target abundance (brightest fluorophores for lowest-expressed targets)

  • Consider compensation requirements for flow cytometry

  • Test antibodies individually before combining

• Staining sequence optimization:

  • Determine optimal order of antibody application

  • Consider sequential staining with intermediate fixation steps

  • Test for potential antibody cross-reactivity

• Acquisition settings:

  • Optimize PMT voltages or exposure times for each channel

  • Set up proper compensation or spectral unmixing

  • Use single-stained controls for each fluorophore

• Analysis approaches:

  • Use appropriate gating strategies for flow cytometry

  • Apply spectral unmixing algorithms for microscopy

  • Quantify co-localization using specialized software

Application examples:

• Co-localization of SLC12A7 with other ion transporters

• Analysis of SLC12A7 expression in specific cell populations identified by lineage markers

• Correlation of SLC12A7 expression with activation or functional markers

What are common technical challenges with FITC-conjugated antibodies and their solutions?

Researchers frequently encounter these challenges when working with FITC-conjugated antibodies:

Critical optimization steps:

• Titrate antibody to determine optimal signal-to-noise ratio

• Test fixation and permeabilization protocols if targeting internal epitopes

• Evaluate multiple blocking reagents to minimize background

• Compare different antigen retrieval methods for tissue sections

• Validate results with complementary techniques

How can researchers accurately quantify SLC12A7 expression using FITC-conjugated antibodies?

For accurate quantification of SLC12A7 expression:

Flow Cytometry Quantification:

• Relative quantification:

  • Calculate median fluorescence intensity (MFI)

  • Determine staining index: (Sample MFI - Isotype control MFI) / Standard deviation of isotype control

  • Report percentage of positive cells above threshold set with isotype control

• Absolute quantification:

  • Use calibration beads with known quantities of FITC molecules

  • Generate standard curve relating fluorescence intensity to molecule number

  • Convert sample fluorescence to molecules of equivalent soluble fluorochrome (MESF)

Microscopy Quantification:

• Image acquisition standardization:

  • Use identical exposure settings across all samples

  • Include fluorescence standards in each imaging session

  • Apply flat-field correction for uniform illumination

• Analysis approaches:

  • Measure integrated density or mean fluorescence intensity of regions of interest

  • Subtract background from areas without specific staining

  • Normalize to cell number using nuclear counterstain

  • Apply threshold-based segmentation for automated analysis

Standardization considerations:

• Always include positive and negative controls

• Maintain consistent instrument settings

• Verify linearity of detection range

• Account for potential autofluorescence

What storage and handling practices ensure optimal performance of FITC-conjugated SLC12A7 antibodies?

Proper storage and handling are critical for maintaining antibody performance:

Storage Recommendations:

• Temperature:

  • Long-term: Store at -20°C in small aliquots

  • Short-term (up to 1 month): Store at 2-8°C

  • Avoid repeated freeze-thaw cycles

• Light protection:

  • Store in amber vials or wrapped in aluminum foil

  • Minimize exposure to light during all handling steps

  • Work under reduced ambient lighting when possible

• Buffer conditions:

  • Store in manufacturer's recommended buffer (typically PBS with stabilizing proteins)

  • Ensure buffer contains appropriate preservatives (typically 0.09% sodium azide)

  • Maintain recommended pH (typically 7.2-7.4)

Handling Best Practices:

• Aliquot stock antibody upon receipt to avoid repeated freeze-thaw cycles

• Centrifuge vial briefly before opening to collect liquid at bottom

• Use clean pipette tips for each handling

• Return to appropriate storage conditions immediately after use

• Document date of receipt, aliquoting, and usage

• Periodically test performance with positive control samples

• Transport on ice and protected from light

How should researchers interpret flow cytometry data obtained with SLC12A7 Antibody, FITC conjugated?

Proper interpretation of flow cytometry data requires systematic analysis:

Gating Strategy:

• Apply FSC/SSC gating to identify intact cells and exclude debris

• Use viability dye to exclude dead cells (critical for accurate interpretation)

• For adherent cells, apply doublet discrimination using FSC-H vs FSC-A

• Set positive/negative boundaries using isotype and unstained controls

Data Analysis Framework:

• Qualitative analysis:

  • Determine if expression pattern is unimodal, bimodal, or multimodal

  • Compare histogram overlays between experimental and control samples

  • Assess shifts in fluorescence intensity relative to controls

• Quantitative analysis:

  • Calculate percent positive cells above threshold

  • Determine median fluorescence intensity (MFI) for positive populations

  • Calculate fold change in MFI relative to controls

  • Apply appropriate statistical tests for group comparisons

Example interpretation table:

This approach allows for standardized reporting of SLC12A7 expression across experiments and samples .

What experimental approaches can researchers use to investigate SLC12A7 function using antibody-based techniques?

Antibody-based techniques offer valuable insights into SLC12A7 function:

Protein Localization Studies:

• Subcellular localization:

  • Use confocal microscopy with FITC-conjugated SLC12A7 antibody and organelle markers

  • Examine redistribution following stimuli or stress conditions

  • Quantify plasma membrane versus intracellular distribution

• Tissue expression mapping:

  • Apply immunohistochemistry across diverse tissue types

  • Correlate expression with physiological function

  • Compare normal versus pathological tissue samples

Functional Studies:

• Co-localization with interaction partners:

  • Combine FITC-SLC12A7 antibody with antibodies against known or suspected interaction partners

  • Quantify degree of co-localization using Pearson's or Mander's coefficients

  • Examine changes in co-localization following experimental manipulations

• Internalization and trafficking studies:

  • Use antibody to track SLC12A7 internalization following stimulation

  • Quantify surface versus internalized protein over time

  • Correlate with functional readouts of ion transport

• Expression correlation with phenotypes:

  • Stratify samples based on SLC12A7 expression levels

  • Correlate with cellular phenotypes or disease markers

  • Develop expression-based classification systems

These approaches can provide significant insights into how SLC12A7 contributes to cellular physiology and potential pathological mechanisms .

How does SLC12A7 relate to disease mechanisms, and how can researchers investigate this relationship?

While the provided search results don't offer specific details about SLC12A7 in disease, researchers can employ several strategies to investigate potential relationships:

Disease Association Studies:

• Expression analysis in disease tissues:

  • Compare SLC12A7 levels between normal and diseased tissues using FITC-conjugated antibody

  • Correlate expression with disease severity or progression

  • Examine subcellular localization changes in disease states

• Genetic correlation studies:

  • Analyze relationship between SLC12A7 genetic variants and protein expression

  • Use antibody-based methods to quantify protein levels in samples with different genotypes

  • Correlate expression with functional outcomes

• Therapeutic response biomarkers:

  • Monitor SLC12A7 expression changes following treatment

  • Determine if baseline expression predicts treatment response

  • Investigate potential as a companion diagnostic marker

Mechanistic Investigations:

• Pathway analysis:

  • Examine how SLC12A7 expression correlates with other components of ion transport pathways

  • Investigate relationship with cell volume regulation mechanisms

  • Study interactions with protein kinases mentioned in functional annotations

• Functional consequence assessment:

  • Correlate SLC12A7 expression levels with ion transport activity

  • Investigate relationship with cell survival under stress conditions

  • Examine impact on cellular processes like migration or proliferation

What emerging research directions involve SLC12A7 antibodies in therapeutic development?

While direct information about SLC12A7 therapeutic development isn't provided in the search results, researchers can consider these emerging approaches based on related research:

Potential Research Directions:

• Antibody-drug conjugate (ADC) development:

  • Similar to the anti-SLC3A2 ADC described in result , researchers could explore SLC12A7 as an ADC target

  • FITC-conjugated antibodies could help screen for antibody clones with optimal internalization properties

  • Flow cytometry with the FITC-conjugated antibody would help determine target expression in potential disease models

• Diagnostic applications:

  • Develop standardized protocols for SLC12A7 detection in clinical samples

  • Establish expression thresholds for disease classification

  • Create multiplex panels incorporating SLC12A7 with other diagnostic markers

• Functional antibody development:

  • Screen for antibodies that modulate SLC12A7 function (agonist or antagonist activity)

  • Investigate potential therapeutic applications of function-modifying antibodies

  • Use FITC-conjugated versions for rapid screening and characterization

• Companion diagnostic development:

  • Establish SLC12A7 as a predictive biomarker for response to specific therapies

  • Standardize detection methods for clinical implementation

  • Develop quantitative thresholds for treatment decisions

By leveraging well-characterized antibodies like the FITC-conjugated SLC12A7 antibody, researchers can accelerate these emerging research directions and potentially uncover new therapeutic opportunities .