FAM83G Antibody, FITC conjugated

What is FAM83G and what cellular functions does it perform?

FAM83G, also known as PAWS1 (Protein Associated with SMAD1), is a member of the FAM83 protein family characterized by a conserved N-terminal domain called DUF1669 . This domain is homologous to the phospholipase D catalytic domain but lacks essential catalytic histidine residues, suggesting it is unlikely to have phospholipase activity .

FAM83G functions include:

• Regulation of the bone morphogenetic protein (BMP) signaling pathway

• Activation of canonical WNT signaling via interaction with CK1α

• Control of cytoskeletal dynamics and cell migration through interaction with CD2AP

• Potential role as an inducer of apoptosis, with its phosphorylation at S356 modulating heat shock protein 27 (HSP27) phosphorylation

The protein has a calculated molecular weight of 91 kDa, though it often appears around 100 kDa on Western blots due to post-translational modifications .

What is the recommended protocol for storing FAM83G antibody, FITC conjugated to maintain optimal activity?

For maximum shelf life and activity of FAM83G antibody, FITC conjugated:

• Store lyophilized antibody at -20°C for up to one year

• The lyophilized antibody remains stable at room temperature for at least one month

• After reconstitution in sterile pH 7.4 0.01M PBS or appropriate antibody diluent, the antibody remains stable for at least two weeks at 2-4°C

• Avoid repeated freeze/thaw cycles as this significantly diminishes antibody activity

For long-term storage of reconstituted antibody, aliquoting is recommended to minimize freeze/thaw cycles. Each aliquot should be stored at -20°C and thawed only once before use.

What are the validated applications and optimal dilutions for FAM83G antibody, FITC conjugated?

Note: Optimal dilutions/concentrations should be determined by each researcher through dilution series experiments as sensitivity can vary based on cell type and experimental conditions .

How should I design experiments to visualize FAM83G subcellular localization using FITC-conjugated antibodies?

For optimal visualization of FAM83G subcellular localization:

Recommended Protocol:

• Cell Preparation:

  • Culture cells on glass coverslips or chambered slides

  • For adherent cells like U2OS, 70-80% confluence is optimal

• Fixation and Permeabilization:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 for 10 minutes

• Blocking and Antibody Incubation:

  • Block with 3-5% BSA or normal serum for 1 hour

  • Incubate with FAM83G antibody, FITC conjugated at 1:50-1:100 dilution overnight at 4°C

  • Wash thoroughly with PBS (3 × 5 minutes)

• Counterstaining:

  • Counterstain nuclei with DAPI (1:1000) for 5 minutes

  • For cytoskeletal visualization, consider phalloidin staining (Alexa 594 conjugated)

• Imaging:

  • Use confocal microscopy for high-resolution localization studies

  • TIRF microscopy is particularly effective for studying interactions between FAM83G/PAWS1, CD2AP, and actin cytoskeleton at the plasma membrane

Key Findings: FAM83G/PAWS1 shows primarily cytoplasmic localization in U-251 MG cells but may have dynamic localization patterns depending on cell type and signaling status.

What controls should be included when working with FAM83G antibody, FITC conjugated?

A robust experimental design requires the following controls:

When using GFP-tagged constructs as positive controls, researchers have successfully employed GFP-PAWS1 (N-terminal tag) or PAWS1-GFP (C-terminal tag) in transfected cells to validate antibody specificity .

How can I investigate the role of FAM83G in cytoskeletal dynamics using FITC-conjugated antibodies?

To study FAM83G's role in cytoskeletal dynamics:

Experimental Approach:

• Live Cell Imaging:

  • Transfect cells with fluorescent markers: GFP-PAWS1, mCherry-CD2AP, RFP-Zyxin (for focal adhesions), and mApple-LifeAct or Emerald-LifeAct (for actin visualization)

  • Use TIRF microscopy to visualize interactions at the plasma membrane

  • Capture time-lapse images at 5-minute intervals over 1-2 hours

• Co-localization Studies:

  • Use FAM83G antibody, FITC conjugated (1:50) alongside phalloidin-Atto 562 for actin staining

  • Analyze co-localization using Pearson's correlation coefficient

• Interaction Analysis:

  • Perform immunoprecipitation using anti-GFP antibodies from cells expressing GFP-PAWS1

  • Probe for CD2AP (a robust interactor of PAWS1) and actin-related proteins

Key Research Finding: Mass spectrometry has identified CD2AP as one of the most robust protein interactors for PAWS1/FAM83G. This interaction is significant as CD2AP plays a crucial role in controlling actin cytoskeletal dynamics and cell migration .

What are the implications of FAM83G mutations in disease, and how can I design experiments to investigate them?

FAM83G mutations have been implicated in diseases including palmoplantar keratoderma (PPK). To investigate these mutations:

Experimental Design Strategy:

• Mutation Analysis:

  • Create mutation constructs (e.g., FAM83GR265P) using site-directed mutagenesis

  • Express wild-type and mutant proteins in appropriate cell models

• Stability Assessment:

  • Treat cells with cycloheximide (100 μg/ml) to inhibit new protein synthesis

  • Monitor protein abundance at specific time points (0, 1, 3, 6, 24h) by immunoblotting

  • Compare degradation rates between wild-type and mutant proteins

• Functional Assays:

  • Analyze interaction with known binding partners (e.g., CK1α) through co-immunoprecipitation

  • Assess WNT signaling activity through Axin2 transcript levels

  • Examine effects on cell migration using wound healing assays

Case Study Results: The FAM83GR265P variant from a palmoplantar keratoderma patient showed:

• Reduced protein stability (70% reduction by 24h after cycloheximide treatment)

• Loss of interaction with CK1α

• Inability to activate WNT signaling

How can I incorporate FAM83G analysis in cancer research studies using FITC-conjugated antibodies?

For cancer research applications focusing on FAM83G:

Multi-faceted Approach:

• Expression Analysis Across Cancer Types:

  • Perform immunofluorescence with FAM83G antibody, FITC conjugated on tissue microarrays

  • Compare expression levels between tumor and adjacent normal tissues

  • Correlate with clinical parameters and survival data

• Co-expression Studies:

  • Design dual-staining protocols with FAM83G antibody, FITC conjugated and markers for:

    • Cell proliferation (Ki-67)

    • Stemness markers

    • Immune cell infiltration markers

• Prognostic Value Assessment:

  • Stratify patients into high/low FAM83G expression groups

  • Perform Kaplan-Meier survival analysis and Cox proportional hazard regression

Pan-Cancer Analysis Findings: FAM83G expression has significant prognostic implications across multiple cancer types. For example:

• High FAM83G expression correlates with poor prognosis in LUAD (Lung Adenocarcinoma) (p = 0.012)

• Low FAM83G expression is associated with better prognosis in LAML (Acute Myeloid Leukemia) (p < 0.001)

The FAM83 family gene expression patterns also correlate with tumor stemness indices (RNAss and DNAss) and immune infiltration, suggesting potential roles in cancer progression and response to therapy .

What are common challenges when using FAM83G antibody, FITC conjugated, and how can I overcome them?

For optimizing signal-to-noise ratio in FAM83G detection, the choice of fixative is critical. Paraformaldehyde (4%) typically yields better results than methanol fixation for preserving FAM83G epitopes and FITC fluorescence.

How can I quantify FAM83G expression levels in different experimental conditions using FITC-conjugated antibodies?

For quantitative analysis of FAM83G expression:

Quantification Protocol:

• Image Acquisition:

  • Capture multiple fields (minimum 5-10) per condition

  • Use identical exposure settings across all samples

  • Include calibration standards if absolute quantification is required

• Image Analysis:

  • Use software like ImageJ/Fiji, CellProfiler, or specialized microscopy software

  • Define regions of interest (ROI) around cells or specific subcellular compartments

  • Measure parameters including:

    • Mean fluorescence intensity

    • Integrated density

    • Cell area

    • Background signal for subtraction

• Data Normalization:

  • Subtract background fluorescence from all measurements

  • Normalize to cell number or area

  • For relative quantification, express as fold change compared to control

• Statistical Analysis:

  • Apply appropriate statistical tests (t-test for two conditions, ANOVA for multiple conditions)

  • Present data as mean ± standard deviation/SEM with significance levels

Application Example: When comparing FAM83G expression between normal and cancer cells, researchers have found significant upregulation in several cancer types. Quantitative IF has revealed that FAM83G expression negatively correlates with stromal score in multiple tumors, indicating that FAM83G expression is associated with higher tumor purity .

How can FAM83G antibody, FITC conjugated be used to study the relationship between FAM83G and WNT signaling?

Recent research has revealed FAM83G's crucial role in WNT signaling. To investigate this:

Experimental Design:

• Co-localization Studies:

  • Use FAM83G antibody, FITC conjugated alongside antibodies against WNT pathway components (β-catenin, CK1α)

  • Perform stimulation experiments with WNT ligands and monitor redistribution patterns

• Functional Analysis in Model Systems:

  • Generate FAM83G knockout and mutant (FAM83GR265P) cell lines

  • Measure WNT pathway activation through Axin2 transcript levels by qRT-PCR

  • Perform TOPFlash reporter assays to quantify WNT transcriptional activity

• Mutation Impact Analysis:

  • Compare wild-type FAM83G with FAM83GR265P variant's ability to restore WNT signaling in FAM83G−/− cells

Key Research Finding: The FAM83GR265P variant from a palmoplantar keratoderma patient demonstrates:

• Reduced protein stability

• Loss of CK1α binding capacity

• Inability to activate WNT signaling

This suggests that the interaction between FAM83G and CK1α is crucial for WNT pathway activation, providing new insights into potential therapeutic targets for WNT-dependent diseases.

What advanced co-localization techniques can be used to study the interaction between FAM83G and its binding partners?

To investigate FAM83G's protein interactions at high resolution:

Advanced Co-localization Techniques:

• Proximity Ligation Assay (PLA):

  • Use FAM83G antibody together with antibodies against interaction partners (e.g., CD2AP, CK1α)

  • Visualize protein interactions with single-molecule sensitivity

  • Quantify interaction events per cell

• Super-Resolution Microscopy:

  • Apply techniques like STORM, PALM, or SIM for nanoscale resolution

  • Resolve co-localization beyond the diffraction limit

  • Combine with FITC-conjugated FAM83G antibodies for multi-color imaging

• Fluorescence Resonance Energy Transfer (FRET):

  • Use FITC-conjugated FAM83G antibody as donor

  • Use antibodies against potential interactors conjugated with compatible acceptor fluorophores

  • Measure energy transfer to confirm protein proximity (<10 nm)

• Live Cell Imaging with TIRF Microscopy:

  • Transfect cells with GFP-PAWS1, mCherry-CD2AP, RFP-Zyxin, and fluorescent actin markers

  • Track dynamic interactions at the cell membrane

  • Capture images at 5-minute intervals for 1-2 hours

Research Application: Using TIRF microscopy, researchers have demonstrated that PAWS1/FAM83G controls cytoskeletal dynamics by influencing the organization of F-actin and focal adhesions and the distribution of the actin stress fiber network . This methodology revealed dynamic interactions between FAM83G and its partners that would be missed by standard confocal microscopy.

By combining these advanced techniques with FITC-conjugated FAM83G antibodies, researchers can gain unprecedented insights into the spatial and temporal dynamics of FAM83G interactions in various cellular contexts.