GREM1 Antibody, HRP conjugated

What is GREM1 and what are its known biological functions?

GREM1 (Gremlin-1) is a secreted glycoprotein that functions as a bone morphogenetic protein (BMP) antagonist. It belongs to the DAN family of BMP antagonists and is also known as CKTSF1B1, DAND2, and DRM. GREM1 has a molecular weight of approximately 20.7 kDa . Biologically, GREM1 plays crucial roles in:

• Embryonic development and organogenesis

• Regulation of cell proliferation, migration, and differentiation

• Modulation of BMP signaling pathways

• Regulation of tissue homeostasis

• Involvement in carcinogenesis and fibrotic processes

Recent research has identified GREM1 as being highly expressed in bladder cancer tissues, with expression levels correlating with tumor stage, grade, and patient prognosis . Additionally, GREM1 has been implicated in metabolic disorders, though anti-GREM1 treatment did not demonstrate efficacy in reducing liver inflammation or fibrosis .

What are the optimal storage conditions for GREM1 Antibody, HRP conjugated?

For GREM1 Antibody, HRP conjugated (e.g., product CSB-PA009892LB01HU), the recommended storage conditions are:

• Upon receipt, store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles, as this can compromise antibody integrity

• The antibody is typically formulated in a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative

For long-term storage, aliquoting the antibody into smaller volumes is advisable to minimize freeze-thaw cycles. Always maintain the cold chain when handling the antibody to preserve its activity.

What applications is GREM1 Antibody, HRP conjugated best suited for?

When selecting a GREM1 antibody for a specific application, verify the validated applications provided by the manufacturer. For the HRP-conjugated version, it's particularly well-suited for direct detection in ELISA without the need for secondary antibodies .

How can I validate the specificity of GREM1 Antibody in my experimental system?

Validating antibody specificity is crucial for obtaining reliable research results. For GREM1 Antibody, consider the following validation approaches:

• Positive and negative controls:

  • Positive control: Use tissues or cell lines known to express GREM1 (such as hepatic stellate cells or myofibroblasts)

  • Negative control: Use tissues or cell lines with minimal GREM1 expression (such as biliary epithelial or sinusoidal endothelial cells)

• Knockdown or knockout verification:

  • Perform siRNA/shRNA knockdown of GREM1 in a positive control cell line

  • Compare antibody detection in wildtype versus GREM1-deficient samples

  • Example: The significant suppression of GREM1 (by 93% in T24 and 85% in 5637 bladder cancer cells) following knockdown provides a good system for validation

• Peptide competition assay:

  • Pre-incubate the antibody with excess recombinant GREM1 protein

  • The specific signal should be significantly reduced or eliminated

• Molecular weight verification:

  • Confirm that the detected band appears at the expected molecular weight (~20.7 kDa) in Western blot analyses

• Multiple antibody approach:

  • Use two different GREM1 antibodies targeting different epitopes

  • Concordant results increase confidence in specificity

What are the recommended protocols for using GREM1 Antibody, HRP conjugated in ELISA?

When using GREM1 Antibody, HRP conjugated in ELISA, consider the following methodological recommendations:

• Sample preparation:

  • For tissue samples: Prepare homogenates in appropriate lysis buffer with protease inhibitors

  • For cell culture supernatants: Centrifuge to remove cellular debris

  • For serum/plasma: Dilute samples appropriately (typically 1:10 to 1:100)

• Coating and blocking:

  • Coat wells with capture antibody or sample (depending on ELISA format)

  • Block with 1-5% BSA or non-fat milk in PBS for 1-2 hours at room temperature

• Antibody dilution:

  • Determine optimal working dilution by titration

  • Typical starting dilution for HRP-conjugated antibodies: 1:1000 to 1:5000

  • Dilute in blocking buffer to reduce background

• Detection and visualization:

  • Use TMB, ABTS, or other HRP substrates for colorimetric detection

  • Add stop solution after appropriate development time

  • Measure absorbance at appropriate wavelength (typically 450nm for TMB)

• Controls:

  • Include standard curve using recombinant GREM1 protein

  • Include blank wells (no sample) to assess background

  • Consider including a positive control sample with known GREM1 content

• Optimization tips:

  • Adjust coating buffer pH to optimize antigen binding

  • Optimize antibody incubation time and temperature

  • Consider adding 0.05% Tween-20 to wash buffers to reduce non-specific binding

How can I investigate GREM1's role in cancer progression using GREM1 Antibody?

GREM1 has been implicated in cancer progression, particularly in bladder cancer. Here's a methodological approach to investigate its role:

• Expression analysis:

  • Use GREM1 Antibody in IHC to assess expression patterns in tumor versus normal tissues

  • Correlate expression with clinical parameters (T stage, N stage, pathological stage)

  • Create tissue microarrays for high-throughput analysis across multiple samples

• Functional studies:

  • Generate stable GREM1 knockdown cell lines using shRNA or CRISPR-Cas9

  • Assess impact on proliferation (using CCK-8 or MTT assays)

  • Evaluate effects on apoptosis (using flow cytometry with Annexin V/PI staining)

  • Investigate migration and invasion capabilities (using transwell or wound healing assays)

• Signaling pathway analysis:

  • Examine PI3K/AKT pathway activation, as GREM1 has been shown to promote bladder cancer progression via this pathway

  • Assess phosphorylation of key signaling molecules (p-AKT, p-mTOR) by Western blot

  • Use pathway inhibitors to confirm causality in observed effects

• EMT marker assessment:

  • Evaluate expression of epithelial markers (E-cadherin) and mesenchymal markers (N-cadherin, Vimentin)

  • Correlate changes with GREM1 expression or manipulation

• In vivo models:

  • Establish xenograft models with GREM1-overexpressing or GREM1-knockdown cancer cells

  • Monitor tumor growth, metastasis, and response to therapeutics

  • Analyze tumor samples for pathway activation and EMT marker expression

What experimental approaches can detect interaction between GREM1 and BMP signaling components?

GREM1 functions as a BMP antagonist, making the study of its interactions with BMP signaling components crucial. Consider these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use GREM1 Antibody to pull down GREM1 and associated proteins

  • Probe for BMP2, BMP4, or BMP7 in the immunoprecipitated complex

  • Alternatively, immunoprecipitate BMP proteins and probe for GREM1

• Proximity ligation assay (PLA):

  • Detect protein-protein interactions in situ in fixed cells or tissues

  • Requires antibodies against GREM1 and BMPs from different species

  • Provides spatial information about interaction sites within cells

• Functional reporter assays:

  • Utilize BMP-responsive element (BRE) luciferase reporter systems

  • Assess how GREM1 manipulation affects BMP-induced luciferase activity

  • Can evaluate effects of GREM1 on different BMP ligands specifically

• Analysis of downstream signaling:

  • Monitor SMAD1/5/8 phosphorylation as indicator of BMP pathway activation

  • Assess how GREM1 overexpression or knockdown affects BMP-induced SMAD phosphorylation

  • Research shows GREM1 overexpression affects expression of BMP-related signaling targets (BMP4, SMAD6, SMAD7)

• RNA-seq or qPCR analysis:

  • Evaluate expression changes in BMP target genes following GREM1 manipulation

  • Focus on genes like ID1, ID2, SMAD6, SMAD7, which are direct BMP targets

  • Analysis can reveal the extent and specificity of GREM1's antagonism toward different BMP ligands

How does GREM1 expression correlate with clinical outcomes in cancer patients?

Based on research findings, particularly in bladder cancer, GREM1 expression correlates significantly with clinical outcomes:

How do I troubleshoot weak or non-specific signals when using GREM1 Antibody, HRP conjugated?

When encountering problems with GREM1 Antibody, HRP conjugated, consider these troubleshooting approaches:

• Weak signal issues:

  • Increase antibody concentration (use titration to determine optimal concentration)

  • Extend incubation time or adjust temperature

  • Enhance detection system (longer substrate development, more sensitive substrate)

  • Check sample preparation (protein degradation, insufficient extraction)

  • Verify storage conditions of antibody (degradation due to improper storage)

• High background or non-specific binding:

  • Increase blocking time or concentration (5% BSA or milk instead of 1-3%)

  • Add 0.05-0.1% Tween-20 to wash buffers and increase washing steps

  • Reduce antibody concentration

  • Pre-absorb antibody with non-specific proteins

  • Filter buffers to remove particulates

• Cross-reactivity concerns:

  • Verify antibody specificity using knockdown/knockout controls

  • Perform peptide competition assays

  • Check for homologous proteins that might cross-react

  • Consider using more specific antibodies if available

• Optimization matrix approach:

  • Systematically vary key parameters (antibody dilution, incubation time, temperature)

  • Document all changes and results to identify optimal conditions

  • Once optimized, maintain consistent protocols

What are the considerations for using GREM1 Antibody in multiplex analyses?

When incorporating GREM1 Antibody into multiplex analyses (detecting multiple targets simultaneously), consider these methodological recommendations:

• Antibody compatibility:

  • Ensure antibodies are from different host species to avoid cross-reactivity

  • If using multiple antibodies from the same species, use directly conjugated antibodies with different fluorophores

  • Test for cross-reactivity between secondary antibodies

• Signal separation:

  • For fluorescent detection, choose fluorophores with minimal spectral overlap

  • For chromogenic detection, ensure clear visual distinction between signals

  • For HRP-conjugated antibodies, consider sequential detection with different substrates

• Panel design considerations:

  • Balance between number of targets and signal clarity

  • Include proper single-stain controls for each antibody

  • Include unstained controls to assess autofluorescence

• Sample preparation:

  • Optimize fixation and permeabilization to accommodate all targets

  • Consider antigen retrieval methods compatible with all targets

  • Use blocking reagents that reduce background for all antibodies in the panel

• Data analysis:

  • Account for spectral overlap through compensation (flow cytometry) or unmixing (imaging)

  • Establish robust gating or thresholding strategies

  • Consider advanced analysis tools for high-dimensional data (tSNE, UMAP)

Which cell types express GREM1 and how does this impact experimental design?

Understanding cell-specific expression of GREM1 is crucial for experimental design. Based on current research:

• Primary cell types expressing GREM1:

  • Hepatic stellate cells and myofibroblasts show high GREM1 expression

  • Fibroblasts in fibrotic areas (particularly THY1/COL3A1-positive cells)

  • Cancer-associated fibroblasts in tumor microenvironment

  • Low or absent expression in biliary epithelial and sinusoidal endothelial cells

• Experimental design implications:

  • Select appropriate cell models based on research question

  • Consider co-culture systems to study interactions between GREM1-producing and GREM1-responsive cells

  • Evaluate primary cells versus cell lines for physiological relevance

• Tissue expression patterns:

  • Higher expression in bladder cancer tissues compared to normal tissues

  • Expression increases with cancer progression (T stage, N stage, pathological stage)

  • Expression in fibrotic areas in metabolic-associated liver diseases

• Cell isolation and purification strategies:

  • FACS sorting based on cell-specific markers to isolate GREM1-expressing cells

  • Laser capture microdissection to isolate specific regions from tissue sections

  • Magnetic bead separation for specific cell populations

• Validation approaches:

  • In situ hybridization to confirm mRNA expression in specific cell types

  • Dual immunofluorescence to co-localize GREM1 with cell-type specific markers

  • Single-cell RNA sequencing to comprehensively map expression across cell types

How can I quantitatively assess GREM1 protein levels in tissue samples?

For quantitative assessment of GREM1 protein in tissue samples, consider these methodological approaches:

• Immunohistochemistry (IHC) quantification:

  • Use digital image analysis software to quantify staining intensity

  • Develop a consistent scoring system (e.g., H-score = percentage × intensity)

  • Include internal controls for normalization across samples

  • Consider automated platforms for higher throughput and objectivity

• Western blot analysis:

  • Standardize protein extraction protocols for tissue homogenates

  • Include loading controls (β-actin, GAPDH) for normalization

  • Use densitometry software for band quantification

  • Create standard curves with recombinant GREM1 for absolute quantification

• ELISA-based quantification:

  • Homogenize tissue samples in appropriate buffer with protease inhibitors

  • Develop standard curves using recombinant GREM1

  • Consider sandwich ELISA for higher specificity

  • Account for matrix effects in tissue homogenates

• Mass spectrometry approaches:

  • Targeted MS approaches for absolute quantification

  • Incorporate labeled internal standards

  • Sample fractionation to reduce complexity

  • Data analysis using specialized software for protein quantification

• Multiplex protein assays:

  • Luminex or similar bead-based assays for simultaneous quantification of multiple proteins

  • Correlate GREM1 levels with other biomarkers or signaling molecules

  • Establish normalization strategies across different protein targets