CRLF1 Antibody, HRP conjugated

What is CRLF1 and what biological functions does it serve?

CRLF1 (cytokine receptor-like factor 1) is a secreted protein of approximately 46 kDa (422 amino acids) belonging to the cytokine receptor family. The protein functions as both a co-ligand with CLCF1 and independently as a regulatory molecule in multiple biological processes. CRLF1 has been implicated in fibrotic processes, signaling pathway modulation, and cancer progression. Research has demonstrated that CRLF1 can promote fibrosis via the ERK signaling pathway at the post-transcriptional level and is required for the pro-fibrotic effect of TGF-β1 . Additionally, CRLF1 enhances cell proliferation and metastasis in papillary thyroid carcinoma through activation of the ERK1/2 and AKT pathways .

How does CRLF1 antibody detection differ between normal and pathological tissues?

In normal tissues, CRLF1 exhibits relatively low basal expression, making detection challenging without sensitive methods. In pathological states, such as ligamentum flavum hypertrophy (HLF), CRLF1 shows significantly upregulated expression. For instance, immunohistochemistry studies have demonstrated that CRLF1 is strongly accumulated around injured kidney tubules in ischemia-reperfusion injury models compared to minimal expression in control tissues . Similarly, in hypertrophic ligamentum flavum, CRLF1-positive cells correlate positively with the number of myofibroblasts (α-SMA-positive cells), suggesting its involvement in fibrotic processes . These expression differences require different antibody dilutions and detection protocols when studying normal versus pathological samples.

What are the key considerations when selecting an HRP-conjugated CRLF1 antibody?

When selecting an HRP-conjugated CRLF1 antibody, researchers should consider several critical factors:

• Reactivity specificity: Confirm the antibody's reactivity with your species of interest. For example, product 17027-1-AP has been validated for human samples .

• Clonality: Polyclonal antibodies like 17027-1-AP offer high sensitivity through recognition of multiple epitopes, while monoclonal antibodies provide better specificity for a single epitope .

• Application compatibility: Verify the antibody has been validated for your specific application (Western blot, IHC, ELISA, etc.). Some antibodies perform differently across applications.

• Epitope location: Consider whether the antibody targets domains relevant to your research question, especially if studying specific CRLF1 interactions or modifications.

• Validation data: Examine the manufacturer's validation data, including positive/negative controls and specificity testing to ensure reliability.

What are the optimal conditions for immunohistochemical detection of CRLF1?

For optimal immunohistochemical (IHC) detection of CRLF1, researchers should consider the following protocol guidelines based on published research:

• Fixation: Formalin-fixed, paraffin-embedded tissues generally provide reliable CRLF1 detection.

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) has been successful in exposing CRLF1 epitopes.

• Blocking: Use 5-10% normal serum from the same species as the secondary antibody for 1 hour at room temperature to minimize background.

• Primary antibody: Dilute CRLF1 antibody (e.g., 17027-1-AP) at 1:100-1:200 and incubate overnight at 4°C for optimal binding .

• Secondary detection: When using HRP-conjugated antibodies, ensure compatible secondary detection systems or direct HRP antibody application with appropriate incubation times (typically 1-2 hours at room temperature).

• Visualization: DAB (3,3′-diaminobenzidine) substrate provides good contrast for CRLF1 detection, with incubation times of 2-10 minutes depending on expression levels.

• Counterstaining: Light hematoxylin counterstaining for 30-60 seconds allows cellular context without obscuring CRLF1 signal.

Studies successfully implementing CRLF1 IHC have detected significant differences between normal and hypertrophic ligamentum flavum tissues, with quantifiable increases in CRLF1-positive cells in diseased tissues .

How should CRLF1 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of CRLF1 antibodies is critical for maintaining their activity and ensuring reproducible experimental results:

• Storage temperature: Store at -20°C for long-term preservation. CRLF1 antibodies like 17027-1-AP remain stable for one year after shipment when properly stored .

• Buffer composition: PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 provides optimal stability for CRLF1 antibody preparations .

• Aliquoting: While some formulations specify that aliquoting is unnecessary for -20°C storage, dividing into single-use aliquots is recommended to prevent freeze-thaw cycles for HRP-conjugated antibodies, which are particularly sensitive to repeated freezing and thawing.

• Working dilution preparation: Prepare working dilutions immediately before use and keep on ice during experimental procedures.

• Avoid contamination: Use sterile technique when handling antibody solutions to prevent microbial contamination.

• Protein stabilizers: Some formulations (such as the 20μl size of 17027-1-AP) contain 0.1% BSA for added stability .

What are the recommended protocols for detecting CRLF1 in protein lysates and serum samples?

For protein lysates (Western blot):

• Sample preparation: Lyse cells or tissues in RIPA buffer containing protease inhibitors.

• Protein quantification: Use BCA or Bradford assay to ensure equal loading.

• SDS-PAGE: Load 20-50μg protein per well on 10-12% gels.

• Transfer: Use PVDF membranes for optimal protein binding.

• Blocking: 5% non-fat milk in TBST for 1 hour at room temperature.

• Primary antibody: Incubate with optimized dilution of CRLF1 antibody (1:500-1:2000) overnight at 4°C.

• Detection: If using HRP-conjugated antibody directly, proceed directly to chemiluminescence detection. Otherwise, use compatible secondary antibody.

• Expected band: CRLF1 should appear at approximately 46 kDa .

For serum samples (ELISA):

• Prepare plates: Coat with capture antibody specific to CRLF1.

• Blocking: Block non-specific binding sites with appropriate buffer.

• Sample application: Apply serum samples (typically diluted 1:5 to 1:20).

• Detection: Apply HRP-conjugated CRLF1 antibody.

• Substrate reaction: Add TMB substrate and incubate at 37°C for approximately 1 hour.

• Measurement: Stop reaction and read optical density at 450nm .

Published studies have successfully measured CRLF1 in mouse serum using double antibody sandwich ELISA methods, with protocols involving 2-hour sample incubation at 37°C followed by biotin-conjugated antibody application and subsequent HRP-avidin detection systems .

How can researchers validate the specificity of CRLF1 antibody in their experimental systems?

Validating CRLF1 antibody specificity is crucial for generating reliable research data. Recommended validation approaches include:

• Positive and negative controls:

  • Use tissues/cells known to express high levels of CRLF1 (e.g., fibrotic tissues, certain cancer cells) as positive controls

  • Include tissues/cells with minimal CRLF1 expression as negative controls

  • Consider genetically modified models (CRLF1 knockout) when available

• siRNA knockdown validation:

  • Transfect cells with CRLF1-specific siRNA and confirm signal reduction

  • Include non-targeting siRNA controls to rule out non-specific effects

  • This approach has been successfully implemented in ligamentum flavum cells to demonstrate CRLF1 antibody specificity

• Recombinant protein competition:

  • Pre-incubate antibody with excess purified CRLF1 protein before application

  • Observe signal reduction/elimination to confirm specific binding

• Multiple antibody comparison:

  • Test different antibodies targeting distinct CRLF1 epitopes

  • Concordant results strengthen confidence in specificity

• Western blot verification:

  • Confirm single band at expected molecular weight (46 kDa)

  • Absence of non-specific bands at unexpected molecular weights

• Cross-species reactivity assessment:

  • Evaluate known CRLF1 species reactivity (e.g., 17027-1-AP is reactive with human samples)

  • Consider evolutionary conservation when interpreting results

How is CRLF1 antibody used to investigate signaling pathway activation?

CRLF1 antibody can be leveraged to investigate multiple signaling pathways through various experimental approaches:

• Co-immunoprecipitation studies:

  • HRP-conjugated CRLF1 antibodies can identify binding partners involved in signaling complexes

  • This approach has helped establish CRLF1's relationship with ERK signaling components

• Western blot for pathway activation markers:

  • After CRLF1 manipulation (overexpression/knockdown), examine phosphorylation status of downstream effectors

  • Research has demonstrated that CRLF1 activates ERK and AKT pathways in multiple cell types, with phosphorylation occurring rapidly after stimulation

• Immunofluorescence co-localization:

  • Dual staining with CRLF1 and pathway components (e.g., phospho-ERK)

  • Researchers have shown co-expression patterns between CRLF1 and α-SMA in fibrotic tissues

• Inhibitor studies:

  • Use pathway-specific inhibitors (e.g., U0126 for MEK/ERK, MK-2206 for AKT) alongside CRLF1 manipulation

  • Research has demonstrated that the oncogenic effects induced by CRLF1 can be suppressed by these inhibitors

• Quantitative assays following stimulation:

  • Treat cells with recombinant CRLF1 and measure changes in pathway activation

  • Previous studies observed that CRLF1 enhanced ligamentum flavum fibrosis via ERK signaling at the post-transcriptional level

These approaches have revealed that CRLF1 significantly impacts both the ERK1/2 and AKT pathways, serving as a critical regulatory node in multiple cellular processes including fibrosis and cancer progression.

What role does CRLF1 play in fibrotic disorders and how can researchers investigate this?

CRLF1 has emerged as a key regulator in fibrotic processes, particularly in ligamentum flavum hypertrophy. Researchers can investigate this role through:

• Expression analysis in fibrotic tissues:

  • Use IHC with CRLF1 antibodies to compare expression between normal and fibrotic tissues

  • Quantify CRLF1-positive cells as demonstrated in studies of hypertrophic ligamentum flavum

  • Implement double immunofluorescence staining for CRLF1 and fibrosis markers (e.g., α-SMA)

• In vitro fibrosis models:

  • Apply CRLF1 recombinant protein to primary cells and assess fibrotic marker expression

  • Measure cell migration using scratch assays to evaluate fibrotic potential

  • Compare CRLF1 effects with established pro-fibrotic factors like TGF-β1

• Pathway dissection:

  • Use siRNA knockdown of CRLF1 to evaluate its necessity in fibrosis development

  • Studies have shown that siCRLF1 significantly reduces the pro-fibrotic effects of inflammatory cytokines and mechanical stress

  • Combine with ERK pathway inhibitors to determine mechanism specificity

• Transcriptional vs. post-transcriptional effects:

  • Analyze both mRNA (RT-qPCR) and protein (Western blot) levels of fibrotic markers

  • Research indicates CRLF1's pro-fibrotic effects operate primarily at the post-transcriptional level, distinguishing it from some other fibrotic mediators

• Animal models:

  • Evaluate CRLF1 expression in mouse models of fibrosis (e.g., bipedal standing posture for ligamentum flavum hypertrophy)

  • Consider CRLF1 overexpression approaches to induce fibrotic phenotypes

These methodologies have established CRLF1 as a critical factor in fibrosis, showing that it enhances myofibroblast transdifferentiation and extracellular matrix production through ERK-dependent mechanisms.

How can CRLF1 antibodies be used to study cancer progression and metastasis?

CRLF1 antibodies provide valuable tools for investigating cancer biology, particularly in relation to progression and metastasis:

• Tumor tissue microarray analysis:

  • Apply CRLF1 antibodies to compare expression across tumor stages and grades

  • Correlate expression with clinical outcomes (survival, metastasis)

  • Studies have shown high CRLF1 levels associated with aggressive clinicopathological features and poor disease-free survival in papillary thyroid carcinoma

• Epithelial-mesenchymal transition (EMT) assessment:

  • Use CRLF1 antibodies alongside EMT markers (E-cadherin, N-cadherin, vimentin)

  • Research has demonstrated that CRLF1 induces EMT in cancer cells

  • Implement immunofluorescence co-staining to visualize EMT progression

• Cell migration and invasion assays:

  • Manipulate CRLF1 expression and measure functional consequences using:

    • Transwell migration assays

    • Scratch wound healing assays

    • 3D invasion models

  • Studies found that CRLF1 increases migration and invasion in papillary thyroid carcinoma cells

• Xenograft tumor models:

  • Use CRLF1 antibodies to monitor expression in tumor xenografts

  • Compare tumor growth rates between CRLF1-overexpressing and control xenografts

  • Evaluate metastatic potential through secondary site analysis

• Mechanism investigation:

  • Apply CRLF1 antibodies in combination with signaling pathway inhibitors

  • Research demonstrated that oncogenic effects of CRLF1 were suppressed by MEK inhibitor U0126 or AKT inhibitor MK-2206

  • Investigate downstream effectors through Western blot and immunoprecipitation

These approaches have established CRLF1 as a potential therapeutic target in cancers like papillary thyroid carcinoma by demonstrating its role in promoting malignant phenotypes through specific signaling pathways.

What techniques can be used to study CRLF1 interactions with CLCF1 and other binding partners?

Investigating CRLF1's interactions with its binding partners requires specialized techniques:

• Co-immunoprecipitation (Co-IP):

  • Use CRLF1 antibody to pull down protein complexes

  • Western blot for potential binding partners (CLCF1, receptors, signaling components)

  • Include appropriate controls (IgG, lysate input)

  • Consider crosslinking for transient interactions

• Proximity ligation assay (PLA):

  • Utilize CRLF1 antibody with antibodies against suspected interaction partners

  • Fluorescent signal indicates proteins in close proximity (<40nm)

  • Particularly valuable for visualizing interactions in situ

• ELISA-based binding assays:

  • Coat plates with recombinant CRLF1 or binding partner

  • Apply HRP-conjugated antibodies to detect complex formation

  • Measure binding kinetics through titration experiments

• Surface plasmon resonance (SPR):

  • Immobilize CRLF1 on sensor chip

  • Measure real-time binding with potential partners

  • Determine association/dissociation constants

• Bimolecular fluorescence complementation (BiFC):

  • Fuse CRLF1 and potential partner with complementary fluorescent protein fragments

  • Interaction brings fragments together, restoring fluorescence

  • Visualize interaction through microscopy

• Functional validation of interactions:

  • Compare CRLF1 alone versus CRLF1-CLCF1

  • Research has indicated CRLF1 may function independently from CLCF1 in certain contexts

  • Studies found that while CRLF1 was upregulated in hypertrophic ligamentum flavum, CLCF1 mRNA was not elevated, suggesting autonomous roles for CRLF1

These techniques can help resolve the ongoing debate about whether CRLF1 primarily functions as a co-ligand with CLCF1 or has independent regulatory functions, which has important implications for therapeutic targeting strategies.

Why might Western blot detection of CRLF1 show inconsistent results, and how can this be addressed?

Inconsistent CRLF1 detection in Western blots can occur for several reasons:

• Protein extraction challenges:

  • CRLF1 is a secreted protein that may be lost during standard extraction

  • Solution: Collect and concentrate cell culture supernatants for secreted CRLF1

  • Use TCA precipitation or commercial protein concentration kits

  • For tissue samples, modify extraction protocols to retain extracellular proteins

• Antibody specificity issues:

  • Different epitopes may be variably accessible in denatured samples

  • Solution: Test multiple antibodies targeting different CRLF1 regions

  • Validate with positive controls (recombinant CRLF1 protein)

  • Consider native vs. reducing conditions

• Post-translational modifications:

  • CRLF1 may undergo glycosylation affecting apparent molecular weight

  • Solution: Use deglycosylation enzymes prior to Western blot

  • Compare observed band with calculated molecular weight (46 kDa)

  • Run gradient gels for better resolution

• Protein-protein complexes:

  • CRLF1 may form stable complexes with CLCF1 or other partners

  • Solution: Include stronger denaturing conditions (increased SDS, heat)

  • Consider non-reducing vs. reducing conditions

  • Use 2D gel electrophoresis for complex samples

• Technical optimization:

  • Solution: Optimize transfer conditions for glycoproteins

  • Extend blocking time to reduce background

  • Test different membrane types (PVDF typically performs better than nitrocellulose for glycoproteins)

  • Consider longer primary antibody incubation (overnight at 4°C)

How can researchers optimize CRLF1 detection in low-expression experimental systems?

Detecting low-abundance CRLF1 requires enhanced sensitivity approaches:

• Signal amplification methods:

  • Use tyramide signal amplification (TSA) for immunohistochemistry

  • Apply enhanced chemiluminescence (ECL) substrates with extended exposure for Western blots

  • Consider biotin-streptavidin amplification systems

• Sample enrichment:

  • Implement immunoprecipitation before Western blot

  • Concentrate secreted proteins from larger culture volumes

  • Use gradient centrifugation for subcellular fractionation

• Alternative detection platforms:

  • Consider more sensitive assays like digital ELISA (Simoa)

  • Utilize droplet digital PCR for transcript detection

  • Apply proximity extension assays for protein quantification

• Technical optimization:

  • Increase antibody concentration while extending incubation time

  • Reduce washing stringency (lower salt, shorter washes)

  • Optimize antigen retrieval for tissue sections (test multiple buffers and pH conditions)

  • Use signal enhancing polymers for IHC/ICC applications

• Positive controls and validation:

  • Include samples with known CRLF1 expression (e.g., cells stimulated with TGF-β1, which significantly increases CRLF1 expression)

  • Use recombinant CRLF1 protein as technical positive control

  • Consider transfection to create overexpression samples for protocol optimization

• Detection system selection:

  • HRP-conjugated antibodies typically offer greater sensitivity than alkaline phosphatase conjugates

  • Fluorescent detection may offer advantages for co-localization studies

  • Consider cooled CCD cameras for digital imaging of low signals

How can researchers interpret contradictory findings regarding CRLF1 function across different experimental systems?

Contradictory findings regarding CRLF1 function may arise from biological complexity and methodological differences:

• Tissue-specific effects:

  • CRLF1 shows context-dependent activities in different tissues

  • Example: CRLF1 inhibits collagen III expression in liver but promotes fibrosis in ligamentum flavum

  • Solution: Carefully define tissue context and avoid generalization across systems

  • Consider testing identical interventions across multiple tissues/cells

• Interaction partner availability:

  • CRLF1 function may depend on presence/absence of binding partners (e.g., CLCF1)

  • Solution: Characterize expression of known binding partners in your system

  • Consider co-expression experiments with potential partners

• Transcriptional vs. post-transcriptional regulation:

  • CRLF1 effects may differ at different regulatory levels

  • Example: Studies showed CRLF1's pro-fibrotic effects operate primarily at post-transcriptional rather than transcriptional level

  • Solution: Analyze both mRNA and protein responses to CRLF1 manipulation

• Signaling pathway context:

  • Downstream effects depend on pre-existing activation state of signaling networks

  • Solution: Characterize baseline pathway activation in your model

  • Consider pathway inhibitor experiments to isolate specific mechanisms

• Experimental approach differences:

  • Knockdown vs. overexpression may reveal different aspects of function

  • Acute vs. chronic manipulation may yield different results

  • Solution: Use complementary approaches (gain and loss of function)

  • Consider time-course experiments to distinguish immediate vs. delayed effects

• Data integration approach:

  • Integrate findings across models using systems biology approaches

  • Solution: Develop computational models incorporating context-dependent interactions

  • Map contradictions to specific biological variables to identify determinants of functional differences

What are the most promising emerging applications for CRLF1 antibodies in biomedical research?

Emerging research directions for CRLF1 antibodies include:

• Biomarker development:

  • CRLF1 shows potential as a biomarker for fibrotic conditions

  • Potential application in cancer prognosis based on association with poor outcomes in PTC

  • Research indicates CRLF1 may be a biomarker linking renal ischemia-reperfusion injury to delayed graft function

  • Consider developing standardized ELISA protocols for clinical application

• Therapeutic targeting:

  • Neutralizing antibodies against CRLF1 may inhibit fibrosis and cancer progression

  • Potential for antibody-drug conjugates targeting CRLF1-expressing cells

  • Opportunity to develop antibodies specifically blocking interaction with particular partners

• Single-cell analysis applications:

  • CRLF1 antibodies can identify specific cellular subpopulations

  • Potential for mass cytometry (CyTOF) applications

  • Integration with spatial transcriptomics for tissue architecture studies

• Pathway crosstalk investigation:

  • CRLF1 interacts with multiple signaling pathways (ERK, AKT)

  • Opportunity to develop multiplexed antibody panels

  • Potential for phospho-specific antibodies to track activation status

• Inflammatory microenvironment characterization:

  • CRLF1 may modulate inflammatory responses

  • Application in immune cell interaction studies

  • Potential role in fibrosis-inflammation feedback loops

• In vivo imaging:

  • Development of labeled antibodies for preclinical imaging

  • Potential for theranostic applications combining imaging and therapy

  • Evaluation of treatment response in animal models

These emerging applications highlight CRLF1's increasing importance as both a research target and potential clinical biomarker or therapeutic target, particularly in fibrotic disorders and certain cancers.

What validation standards should researchers apply when publishing CRLF1 antibody-based findings?

To ensure reproducibility of CRLF1 antibody-based research, authors should adhere to these validation standards:

• Antibody identification and characterization:

  • Report complete antibody information (vendor, catalog number, lot, clone for monoclonals)

  • Specify antibody type (polyclonal, monoclonal, host species)

  • Document target antigen/epitope information when available

  • Example: Polyclonal rabbit anti-CRLF1 (Proteintech, 17027-1-AP), targeting CRLF1 fusion protein Ag10382

• Validation experiments:

  • Demonstrate antibody specificity using positive and negative controls

  • Include knockout/knockdown validation when feasible

  • Document expected molecular weight (46 kDa for CRLF1)

  • Present validation data in supplementary materials

• Experimental conditions:

  • Detail complete protocols including:

    • Sample preparation methods

    • Antibody concentrations/dilutions

    • Incubation conditions (time, temperature)

    • Detection systems

  • Specify any modifications to manufacturer's recommended protocols

• Reproducibility measures:

  • Report number of experimental replicates

  • Include appropriate statistical analyses

  • Consider biological replicates across different samples/conditions

  • Document lot-to-lot testing if performed

• Limitations disclosure:

  • Acknowledge specific limitations of antibody performance

  • Address potential cross-reactivity issues

  • Discuss concordance/discordance with other detection methods

  • Note species reactivity limitations (e.g., human-specific)

These standards align with broader reproducibility initiatives in antibody-based research and help establish confidence in published CRLF1 findings.

How should researchers design experiments to address conflicting literature on CRLF1 function?

When addressing conflicting literature on CRLF1 function, researchers should design experiments with these principles:

• Systematic replication approach:

  • Directly replicate key contradictory findings using identical conditions

  • Systematically vary individual parameters to identify sources of variation

  • Include positive controls from both contradictory findings

• Multi-method validation:

  • Apply complementary methodologies to the same biological question

  • For example, combine genetic manipulation (siRNA, CRISPR) with pharmacological approaches

  • Verify findings across different detection platforms (Western blot, IHC, qPCR)

• Comprehensive model testing:

  • Test hypotheses across multiple relevant cell types/tissues

  • Example: Compare CRLF1 effects in fibroblasts, epithelial cells, and immune cells

  • Include primary cells alongside cell lines when possible

• Context-dependent analysis:

  • Systematically manipulate potential contextual factors:

    • Growth conditions (serum levels, confluency)

    • Cytokine environment

    • Extracellular matrix composition

  • Determine if contradictions are explained by specific contextual variables

• Interaction partner assessment:

  • Evaluate CRLF1 function both independently and in complex with CLCF1

  • Manipulate expression of known binding partners

  • Consider previously unidentified interactions through unbiased approaches

• Time-course resolution:

  • Examine both immediate and delayed responses

  • Distinguish between transient signaling and sustained functional changes

  • Consider developmental timing in in vivo models

• Dose-response relationships:

  • Test across concentration ranges spanning contradictory reports

  • Identify potential biphasic responses or threshold effects

  • Consider physiological versus supraphysiological concentrations