GREM1 Antibody, FITC conjugated

What is the optimal protocol for generating FITC-labeled GREM1 for cellular uptake studies?

Based on recent methodologies, FITC-labelled recombinant human (rh) GREM1 can be generated using a FITC Conjugation Kit (such as Abcam Cat. No: ab102884). The standard procedure involves:

• Starting with recombinant human GREM1 (e.g., R&D Systems, Cat. No: 555190-GR-050)

• Using the conjugation kit according to manufacturer instructions

• Resulting in approximately 183 μg/ml solution of GREM1-FITC

For experimental applications, GREM1-FITC should be directly diluted into cell culture medium with concentrations typically ranging between 0.5-1 μg/mL. The conjugates should be stored at 4°C and protected from light prior to imaging .

How should researchers optimize the visualization of GREM1-FITC in different cell lines?

For optimal visualization of GREM1-FITC across different cell lines:

• Incubation time varies significantly - while binding to cell membranes becomes evident between 5-15 minutes, complete internalization requires 16-24 hours

• Use appropriate cell density (typically sparse to medium density for clear visualization)

• For fixed cells: wash with PBS, fix and counterstain nuclei with DAPI (1 µg/mL in PBS for 20 min)

• For live imaging: conduct in serum-free medium using appropriate imaging platforms (e.g., Nikon 6D live cell imaging microscope)

• Employ confocal microscopy at 40x magnification for detailed subcellular localization studies

Note that different cell lines exhibit varying GREM1 uptake dynamics - HeLa and HCT116 cells show excellent internalization, while other cell types may require optimization .

What are the recommended storage conditions for GREM1 Antibody, FITC conjugated reagents?

For maximum stability and performance:

• Aliquot the antibody upon receipt to avoid repeated freeze/thaw cycles

• Store at -20°C in a non-frost-free freezer

• Protect from light exposure during storage and handling

• Buffer composition (0.01 M PBS, pH 7.4, containing 0.03-0.05% Proclin-300, 50% glycerol) is optimal for maintaining activity

• Avoid prolonged exposure to room temperature

Following these guidelines maintains antibody activity for approximately 12 months from date of receipt .

How can researchers distinguish between membrane-bound and internalized GREM1-FITC in experimental studies?

Differentiating between membrane-bound and internalized GREM1-FITC requires precise methodological approaches:

• Time-course imaging analysis:

  • At 5-15 minutes: initial binding to cell membranes

  • At 60 minutes: distinctive ring-like membrane-bound pattern

  • At 3-6 hours: beginning of internalization

  • At 16-24 hours: predominantly intracellular localization

• Confocal microscopy techniques:

  • Membrane-bound GREM1-FITC: Ring-like pattern at cell periphery

  • Internalized GREM1-FITC: Punctate, perinuclear pattern with localization in cellular extensions

• Endocytosis inhibitor controls:

  • Use PitStop2 (clathrin inhibitor) or Dyngo4a (dynamin inhibitor) to block internalization

  • Compare treated vs. untreated cells to quantify membrane-bound fraction

Quantification indicates approximately 40% decrease in GREM1-FITC uptake with clathrin inhibition and ~80% reduction with dynamin inhibition, suggesting both pathways contribute to GREM1 internalization .

What methodologies can be used to investigate the subcellular localization of internalized GREM1-FITC?

To precisely characterize GREM1-FITC subcellular localization, implement the following methodological approach:

• Co-localization studies with organelle markers:

  • Mitochondria: MitoTracker (200 nM)

  • Endoplasmic reticulum: anti-Calreticulin antibodies

  • Golgi apparatus: anti-GM130 or anti-giantin antibodies

  • Lysosomes: anti-LAMP1 antibodies or cresyl violet staining

  • Early endosomes: EEA1 staining

  • Recycling endosomes: Rab11 staining

• Confocal microscopy protocol:

  • Seed cells on appropriate slides (e.g., Ibidi µ-Slides)

  • Treat with 1 μg/mL GREM1-FITC overnight in complete medium

  • Counterstain with organelle markers (30 min at 37°C)

  • Nuclear staining with DAPI

  • Image at 40x magnification using confocal microscopy (e.g., Leica SP5)

• Analysis findings:

  • No significant co-localization with mitochondria or ER

  • Minimal staining in Golgi apparatus

  • Partial localization to early endosomal compartment

  • Some overlap with lysosomal markers

  • Distinct punctate, perinuclear pattern with staining in cellular extensions and contact points between cells

This comprehensive approach has revealed that internalized GREM1 primarily localizes to early endosomal compartments rather than other organelles .

What experimental controls should be included when studying GREM1-FITC internalization pathways?

A robust experimental design for GREM1-FITC internalization studies requires the following controls:

• Negative controls:

  • BSA-FITC treatment (non-specific protein control)

  • Untreated cells (autofluorescence control)

  • Secondary antibody-only controls for immunofluorescence

• Functional controls:

  • GREM1-FITC vs. non-labeled GREM1 in BMP2-mediated SMAD1/5/8 phosphorylation inhibition assays

  • Verification that FITC labeling does not alter biological activity

• Pathway inhibition controls:

  • Clathrin-mediated endocytosis: PitStop2 (10-50 μM)

  • Caveolin-mediated endocytosis: Dyngo4a (30 μM)

  • Temperature control: 4°C vs. 37°C incubation

• Time-course controls:

  • Short-term exposure (5-60 min) vs. long-term exposure (3-24h)

  • Regular time-point sampling for kinetic analyses

• Cell-type controls:

  • Compare multiple cell lines (HeLa, HCT116, HEK293)

  • Include primary cells when possible

Including these controls enables reliable differentiation between specific and non-specific uptake mechanisms and confirms that both clathrin and caveolin-mediated endocytosis pathways contribute to GREM1 internalization, with caveolin pathways playing a more prominent role .

How does GREM1-FITC internalization differ between normal and cancer cells, and what are the methodological considerations?

Significant differences in GREM1-FITC internalization between normal and cancer cells have been observed, with important methodological considerations:

• Cell-type specific differences:

  • Cancer cells (especially ER-negative breast cancer cells like MDA-MB-468, MDA-MB-453, SKBR3) show elevated GREM1 expression and enhanced uptake

  • Normal epithelial cells (MCF-10A) demonstrate lower baseline internalization

• Experimental design for comparative studies:

  • Use matched cell lines (e.g., MCF-10A vs. MCF-10A-GREM1 overexpressing cells)

  • Standardize cell density, passage number, and culture conditions

  • Normalize GREM1-FITC concentration (typically 0.5-1 μg/mL)

  • Control for differences in membrane composition and endocytic pathway activity

• Quantification approaches:

  • Flow cytometry for population-level quantification

  • Automated image analysis of confocal microscopy for spatial distribution

  • Western blotting of cellular fractions for biochemical verification

• Biological significance:

  • Elevated GREM1 in ER-negative breast cancer correlates with poor prognosis

  • 45.83% (22/48) of ER-negative cell lines show upregulated GREM1 expression

  • Secreted GREM1 levels are significantly higher in cancer cells compared to normal cells

These methodological approaches reveal that cancer cells, particularly ER-negative breast cancer cells, show enhanced GREM1 internalization that correlates with disease progression and prognosis .

What are the optimal methods for studying GREM1 recycling and resecretion in cellular models?

To investigate GREM1 recycling and resecretion, researchers should implement this methodological workflow:

• Conditioned media preparation protocol:

  • Transfect cells (e.g., HEK293T) and culture for 24-48h

  • Wash cells once with PBS

  • Add serum-free medium for 5h at 37°C

  • Centrifuge at 1,200 rpm (13,684 x g) for 10 min to remove debris

  • Either apply to fresh cells immediately or snap-freeze in liquid nitrogen and store at -80°C

• Recycling and resecretion experimental design:

  • Treat first set of cells with GREM1-FITC for internalization

  • Collect conditioned medium after sufficient internalization time

  • Apply this conditioned medium to fresh untreated cells

  • Image recipient cells for GREM1-FITC uptake

• Co-localization with recycling pathway markers:

  • Stain for Rab11 (recycling endosome marker)

  • Assess overlap with internalized GREM1-FITC

• Quantification methods:

  • Measure GREM1-FITC fluorescence intensity in recipient cells

  • Compare with direct GREM1-FITC treatment controls

How can researchers effectively investigate the relationship between GREM1 and BMP signaling pathways using FITC-conjugated tools?

To effectively study GREM1-BMP pathway interactions using FITC-conjugated tools:

• Experimental design for BMP antagonism studies:

  • Pre-treat cells with GREM1-FITC (0.5-1 μg/mL)

  • Challenge with BMP2 (200 ng/mL)

  • Assess changes in SMAD1/5/8 phosphorylation via Western blot

  • Compare with non-labeled GREM1 controls

• Gene expression analysis methodology:

  • Design targeted qPCR panels for BMP-related genes:

    • BMP4, BMP7, SMAD1/6/7

    • INHBB and pathway targets

  • Use GREM1 overexpression systems and/or recombinant GREM1-FITC

  • Include appropriate housekeeping gene controls

• Impact on gene expression findings:

  • GREM1 overexpression reduces BMP4, SMAD6 and SMAD7 expression

  • Significant downregulation of INHBB expression (p = 2.4×10^-5)

  • Upregulation of BMP7 and SMAD1 (p = 0.008 and p = 0.007)

• Functional verification methods:

  • SMAD-responsive luciferase reporter assays

  • Target gene expression profiling

  • Cell proliferation and invasion assays

These experimental approaches reveal that GREM1-FITC is functionally equivalent to unlabeled GREM1 in its ability to inhibit BMP2-mediated SMAD1/5/8 phosphorylation, making it an excellent tool for visualizing GREM1's role in BMP antagonism while simultaneously tracking its cellular localization .

What are common challenges in GREM1-FITC experimental design and how can they be addressed?

Researchers frequently encounter these challenges when working with GREM1-FITC:

• Low signal intensity issues:

  • Cause: Insufficient internalization time or degradation of fluorophore

  • Solution: Extend incubation to 16-24h; prepare fresh conjugates; protect from light

• High background fluorescence:

  • Cause: Non-specific binding or autofluorescence

  • Solution: Include BSA-FITC controls; optimize washing steps; use phenol red-free media

• Variable internalization across cell types:

  • Cause: Differential expression of uptake receptors

  • Solution: Standardize cell density and passage number; characterize baseline internalization for each cell line

• Co-localization analysis difficulties:

  • Cause: Overlapping emission spectra or inadequate image resolution

  • Solution: Use sequential scanning; increase image resolution; apply appropriate co-localization algorithms

• Photobleaching during live imaging:

  • Cause: Prolonged or high-intensity excitation

  • Solution: Reduce laser power; use anti-fade reagents; capture images at defined intervals

• Inconsistent GREM1-FITC activity:

  • Cause: Variable conjugation efficiency or protein denaturation

  • Solution: Verify biological activity via BMP2-mediated SMAD1/5/8 phosphorylation inhibition assays

Most critical is ensuring sufficient incubation time, as GREM1 internalization is notably slow, with binding occurring within minutes but complete internalization requiring 16-24 hours .

How should researchers optimize FITC-conjugated GREM1 antibodies for different experimental applications?

Optimization strategies for FITC-conjugated GREM1 antibodies across applications:

Additional optimization guidelines:

• Antibody selection considerations:

  • Polyclonal antibodies offer higher sensitivity but potentially lower specificity

  • Select antibodies raised against specific regions (e.g., N-terminal or C-terminal)

  • Verify reactivity for your species of interest (e.g., human)

• Performance verification:

  • Always include positive and negative controls

  • Validate with recombinant GREM1 protein

  • Consider blocking peptide controls for specificity confirmation

• Storage optimization:

  • Aliquot upon receipt to minimize freeze-thaw cycles

  • Store at -20°C protected from light

  • Use appropriate buffer (0.01 M PBS, pH 7.4, containing 0.05% Proclin-300, 50% glycerol)

For each application, researchers should perform a titration series to determine the optimal concentration for their specific experimental system .

How can researchers differentiate between specific GREM1-FITC uptake and non-specific cellular internalization?

To distinguish between specific and non-specific GREM1-FITC uptake, implement these methodological controls:

• Essential control experiments:

  • Parallel BSA-FITC treatment (non-specific protein control)

  • Dose-dependent competition with unlabeled GREM1

  • Comparison across multiple cell types with varying GREM1 receptor expression

  • Pre-treatment with receptor blocking antibodies (when receptors identified)

• Temporal analysis approach:

  • Specific GREM1-FITC uptake follows distinctive kinetics:

    • Membrane binding (5-15 min)

    • Ring-like pattern (60 min)

    • Internalization beginning (3-6h)

    • Complete internalization (16-24h)

  • Non-specific uptake typically shows different temporal patterns

• Pathway inhibition strategy:

  • Specific uptake shows sensitivity to endocytosis inhibitors:

    • ~40% reduction with PitStop2 (clathrin inhibitor)

    • ~80% reduction with Dyngo4a (dynamin inhibitor)

  • Non-specific uptake may show different inhibitor sensitivity profiles

• Subcellular localization patterns:

  • Specific GREM1-FITC uptake shows:

    • Punctate, perinuclear pattern

    • Localization in cellular extensions and contact points

    • Partial co-localization with early endosomal markers

  • Non-specific uptake typically shows diffuse patterns

The combination of these approaches enables reliable differentiation between specific receptor-mediated GREM1 internalization and non-specific uptake mechanisms .

How can GREM1-FITC be utilized to study the relationship between GREM1 expression and cancer progression?

GREM1-FITC provides valuable methodological approaches for investigating GREM1's role in cancer:

• Patient-derived xenograft (PDX) models:

  • Inject GREM1-FITC into established PDX models

  • Track distribution and cellular uptake in tumors vs. normal tissues

  • Correlate with clinical outcomes in the original patients

• Ex vivo tissue explant culture protocol:

  • Obtain fresh tumor and adjacent normal tissue from surgical specimens

  • Culture in specialized media with GREM1-FITC (1 μg/mL)

  • Assess differential uptake between normal and tumor cells

  • Analyze downstream molecular changes

• Analysis of prognostic value:

  • GREM1 overexpression correlates with poor outcomes in ER-negative breast cancer

  • Hazard ratio for survival = 1.77 (95% CI: 0.99–3.14, P = 0.05)

  • Specific cellular localization patterns may provide additional prognostic information

• Molecular mechanisms assessment:

  • GREM1 enhances ERRα signaling in breast cancer

  • GREM1 directly activates EGFR, an upstream regulator of ERRα

  • Creates a positive feedback loop that promotes cancer progression

These approaches have revealed that GREM1 is significantly elevated in invasive breast carcinoma compared to normal tissues, with particular relevance to ER-negative breast cancer progression .

What methodologies can researchers employ to investigate the relationship between GREM1 and hereditary cancer syndromes?

To study GREM1's role in hereditary cancer syndromes:

• Genetic analysis approaches:

  • PCR amplification of the 5′ regulatory region of GREM1

  • Detection of the specific duplication associated with hereditary mixed polyposis syndrome

  • Particularly important in Ashkenazi Jewish populations with family history

• Functional assays with GREM1-FITC:

  • Compare GREM1-FITC uptake in cells derived from patients with or without the GREM1 duplication

  • Analyze changes in BMP signaling using SMAD phosphorylation assays

  • Assess polyp formation potential using 3D organoid models

• Clinical correlation methods:

  • Track polyp development in patients with GREM1 mutations

  • Characterize polyp pathology (mixed pathology is characteristic)

  • Assess colorectal cancer risk stratification

• Mechanistic investigation:

  • BMP pathway analysis in carriers vs. non-carriers

  • Transcriptional profiling of colonic tissues

  • Intestinal stem cell behavior alterations

Research indicates that specific duplications in the 5′ regulatory region of the GREM1 gene are found in Ashkenazi Jewish individuals with hereditary mixed polyposis syndrome, characterized by multiple polyps of mixed pathology and high colorectal cancer risk. These approaches enable mechanistic understanding of how GREM1 alterations drive cancer development .

How can advanced imaging techniques enhance GREM1-FITC studies in complex tissue environments?

Advanced imaging methodologies for GREM1-FITC in complex tissues:

• Light sheet fluorescence microscopy (LSFM):

  • Superior for 3D imaging of thick tissue samples

  • Reduced photobleaching compared to confocal microscopy

  • Protocol: Clear tissues using CLARITY or iDISCO+, then image GREM1-FITC distribution

  • Enables visualization of GREM1 transport between cell types (e.g., from fibroblasts to epithelial cells)

• Intravital microscopy approaches:

  • Real-time imaging of GREM1-FITC in living animal models

  • Surgical implantation of imaging windows

  • Combined with transgenic reporter models for cell-type identification

  • Reveals dynamic GREM1 trafficking in physiological context

• Super-resolution techniques:

  • Structured illumination microscopy (SIM): ~100 nm resolution

  • Stimulated emission depletion (STED): ~30-80 nm resolution

  • Single-molecule localization microscopy: ~20 nm resolution

  • Protocol: Traditional sample preparation with thinner sections and specialized mounting media

• Correlative light and electron microscopy (CLEM):

  • Combines fluorescence imaging of GREM1-FITC with ultrastructural EM analysis

  • Protocol: Record fluorescence signal, then prepare the same sample for EM

  • Precisely localizes GREM1 to specific subcellular structures at nanometer resolution

These advanced techniques have revealed that fibroblasts in the muscularis layer of the intestine express GREM1 mRNA that is translated to GREM1 protein, which is then secreted and taken up by proximal epithelial cells - a mechanism potentially involved in both normal tissue homeostasis and cancer development .

What are the most promising approaches for studying GREM1's role in metabolic diseases using FITC-conjugated tools?

Emerging methodologies for investigating GREM1 in metabolic contexts:

• Multi-omics integration approach:

  • Combine GREM1-FITC cellular localization with:

    • Transcriptomics (RNA-seq)

    • Proteomics (mass spectrometry)

    • Metabolomics (LC-MS/MS)

  • Correlate GREM1 uptake patterns with molecular signatures

  • Identify metabolic pathways influenced by GREM1

• Metabolic flux analysis:

  • Treat cells with GREM1-FITC followed by isotope-labeled metabolites

  • Track changes in metabolic pathways using mass spectrometry

  • Correlate with GREM1 internalization patterns

  • Reveal direct metabolic consequences of GREM1 signaling

• In vivo metabolic phenotyping:

  • Administer GREM1-FITC to metabolic disease models

  • Use whole-body imaging to track distribution

  • Correlate with metabolic parameters (glucose tolerance, insulin sensitivity)

  • Tissue-specific analysis of GREM1 uptake and metabolic changes

• Gene expression alterations in metabolic tissues:

  • GREM1 overexpression affects BMP signaling targets:

    • Reduced BMP4, SMAD6 and SMAD7 expression

    • Downregulation of INHBB expression (p = 2.4×10^-5)

    • Upregulation of BMP7 and SMAD1 (p = 0.008 and p = 0.007)

These approaches position GREM1 as a potential therapeutic target in metabolic diseases through its modulation of BMP signaling pathways, which play critical roles in metabolism and energy homeostasis .

How can single-cell analysis techniques be integrated with GREM1-FITC studies?

Integration of single-cell technologies with GREM1-FITC research:

• Single-cell GREM1-FITC uptake analysis:

  • Flow cytometry with high-dimensional analysis (20+ parameters)

  • FACS isolation of cells based on GREM1-FITC uptake levels

  • Correlation with cell-surface markers and functional readouts

  • Reveals heterogeneity in GREM1 responsiveness within populations

• Single-cell RNA sequencing workflow:

  • Treat cell populations with GREM1-FITC

  • FACS-sort based on GREM1-FITC internalization (high vs. low)

  • Perform scRNA-seq on sorted populations

  • Identify transcriptional signatures associated with GREM1 uptake

  • Protocol: 10x Genomics Chromium platform or Smart-seq2

• Spatial transcriptomics approach:

  • Apply GREM1-FITC to tissue sections

  • Image GREM1-FITC localization

  • Perform spatial transcriptomics (Visium or Slide-seq)

  • Correlate GREM1 uptake with spatial gene expression patterns

• Mass cytometry (CyTOF) integration:

  • Develop metal-tagged GREM1 probes

  • Combine with antibodies against signaling molecules

  • Analyze up to 40 parameters simultaneously

  • Map GREM1 uptake to specific cell states and signaling pathways

These integrated approaches can reveal how individual cells within heterogeneous populations respond differently to GREM1, providing insight into cell-specific functions of this important signaling molecule .

What are the methodological considerations for developing GREM1-FITC as a diagnostic tool for cancer detection?

For developing GREM1-FITC as a cancer diagnostic tool, researchers should consider:

• Diagnostic performance assessment protocol:

  • Prepare GREM1-FITC at optimal concentration (0.5-1 μg/mL)

  • Apply to patient-derived samples (tissue sections, circulating tumor cells, liquid biopsies)

  • Compare uptake patterns between cancer and normal cells

  • Calculate sensitivity, specificity, PPV, and NPV for various cancer types

  • Current findings: Significantly elevated in invasive or ductal breast carcinoma in situ compared to normal tissues

• Cancer-specific uptake profiling:

  • Establish baseline GREM1-FITC uptake across normal tissues

  • Create uptake profiles for different cancer types:

    • Breast cancer (ER-positive vs. ER-negative)

    • Colorectal cancer (sporadic vs. hereditary forms)

    • Other cancer types with altered GREM1 expression

  • Quantify uptake differences using standardized metrics

• Clinical sample protocol optimization:

  • Fresh tissue: Immediate processing in GREM1-FITC solution

  • FFPE samples: Deparaffinization and antigen retrieval before GREM1-FITC application

  • Liquid biopsies: Isolation of circulating tumor cells followed by GREM1-FITC treatment

  • All methods require standardized controls and calibration samples

• Multimodal diagnostic approach:

  • Combine GREM1-FITC with other diagnostic markers

  • Develop scoring systems incorporating:

    • Internalization patterns

    • Subcellular localization

    • Quantitative uptake measurements

    • Molecular subtype information