KLB Antibody

What is KLB and what is its biological significance?

KLB, also known as beta-Klotho, is a single-pass type III membrane protein that functions as a co-receptor for multiple endocrine hormones . It plays a critical role in metabolic regulation, particularly in energy balance and glucose metabolism . KLB contributes to the transcriptional repression of cholesterol 7-alpha-hydroxylase (CYP7A1), which is the rate-limiting enzyme in bile acid synthesis . Additionally, it increases the ability of fibroblast growth factor receptors FGFR1 and FGFR4 to bind FGF21 , making it a central player in the FGF signaling pathway.

The importance of KLB in research stems from its involvement in mediating the effects of hormones such as FGF21 and FGF19, highlighting its significance in studies on obesity, diabetes, and other metabolic diseases . Its role as a co-receptor makes it a valuable target for investigating hormonal signaling mechanisms and potential therapeutic interventions.

What applications are KLB antibodies most commonly used for?

KLB antibodies are primarily used in the following research applications:

• Western Blot (WB): KLB antibodies can be used for detection at approximately 1 μg/mL concentration, with HRP-conjugated secondary antibody diluted at 1:50,000-100,000 . This technique allows researchers to confirm the presence and semi-quantify the amount of KLB protein in tissue or cell lysates.

• Enzyme-Linked Immunosorbent Assay (ELISA): KLB antibodies can be used at dilutions around 1:12500 for ELISA applications , enabling quantitative analysis of KLB protein levels in various samples.

• Immunohistochemistry (IHC): Some KLB antibodies are validated for IHC applications , allowing researchers to visualize the distribution and localization of KLB in tissue sections.

These applications enable researchers to investigate KLB expression patterns, protein interactions, and functional changes in various experimental models and clinical samples.

What species reactivity is available for commercial KLB antibodies?

Most commercially available KLB antibodies demonstrate reactivity across multiple species, which is crucial for comparative studies and translational research. Based on the current information:

This cross-reactivity makes these antibodies versatile tools for research across different model systems. When selecting an antibody, researchers should verify that the specific epitope recognized by the antibody is conserved across the species of interest to ensure reliable results.

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

Proper storage and handling of KLB antibodies are essential for maintaining their specificity and sensitivity. Based on manufacturer recommendations:

• KLB antibodies are typically supplied in lyophilized form in PBS buffer with 2% sucrose .

• For reconstitution, add the specified volume of distilled water (typically 50 μL) to achieve a final antibody concentration of 1 mg/mL .

• After reconstitution, aliquot the antibody solution into multiple small volumes to avoid repeated freeze-thaw cycles.

• Store antibody aliquots at -20°C or below for long-term storage .

• Avoid multiple freeze-thaw cycles as they can lead to protein denaturation and loss of antibody activity.

• When working with the antibody, keep it on ice and return to storage promptly after use.

Following these guidelines will help maintain antibody performance and extend its usable lifetime for your experiments.

How can I validate the specificity of a KLB antibody for my experimental system?

Validating antibody specificity is crucial for ensuring reliable experimental results. For KLB antibodies, consider the following validation approaches:

• Positive Control Testing: Use cells or tissues known to express KLB at high levels. For KLB antibodies, liver tissue (rat liver) and certain cell lines (LO2, SGC-7901) have been identified as positive controls .

• Western Blot Analysis: Confirm that the antibody detects a protein of the expected molecular weight for KLB. Look for a single predominant band at the predicted size.

• Blocking Peptide Competition: Pre-incubate the antibody with the immunizing peptide (if available) before application. This should significantly reduce or eliminate specific binding if the antibody is truly specific.

• Knockdown/Knockout Verification: If possible, test the antibody in samples where KLB has been knocked down or knocked out. The signal should be correspondingly reduced or absent.

• Multiple Antibody Comparison: Use two or more antibodies targeting different epitopes of KLB and compare their staining patterns. Concordant results support specificity.

For KLB specifically, the immunogen information (e.g., "Recombinant fusion protein containing a sequence corresponding to amino acids 720-990 of human KLB (NP_783864.1)" ) can help determine if the antibody will recognize your protein of interest in your specific application.

What are the optimal sample preparation methods for KLB detection in different experimental contexts?

Sample preparation significantly impacts KLB antibody performance across different applications:

For Western Blot:

• Use RIPA buffer supplemented with protease inhibitors for cell/tissue lysis.

• Include phosphatase inhibitors if investigating KLB phosphorylation status.

• Denature samples at 95°C for 5 minutes in reducing sample buffer.

• Load 20-50 μg of total protein per lane, depending on KLB expression levels.

For Immunohistochemistry:

• Fix tissues in 4% paraformaldehyde or 10% neutral buffered formalin.

• Consider antigen retrieval methods (heat-induced or enzymatic) to expose epitopes.

• Block endogenous peroxidases and non-specific binding sites before antibody incubation.

• For KLB, optimize incubation times based on tissue type and fixation conditions.

For ELISA:

• Prepare standard curves using recombinant KLB protein.

• Dilute samples appropriately to ensure readings fall within the linear range of detection.

• Include proper negative and positive controls to validate assay performance.

Regardless of application, maintain samples at 4°C during preparation and avoid repeated freeze-thaw cycles to preserve protein integrity and antigenicity.

How do I troubleshoot weak or inconsistent signals when using KLB antibodies?

When encountering weak or inconsistent signals with KLB antibodies, systematically troubleshoot using these approaches:

For Western Blot Issues:

• Increase protein loading (50-100 μg total protein).

• Optimize antibody concentration—try a range around the recommended 1:500 - 1:2000 dilution .

• Extend primary antibody incubation time (overnight at 4°C).

• Use more sensitive detection systems (enhanced chemiluminescence substrates).

• Reduce washing stringency or duration if signal is completely absent.

For Immunohistochemistry Issues:

• Test multiple antigen retrieval methods (citrate buffer, EDTA, or enzymatic).

• Increase antibody concentration or incubation time.

• Use amplification systems like tyramide signal amplification.

• Verify tissue fixation conditions aren't excessively damaging the epitope.

General Troubleshooting:

• Check antibody expiration date and storage conditions.

• Verify that the antibody recognizes the specific isoform or post-translational modification of KLB in your samples.

• Include positive control samples where KLB is known to be expressed, such as liver tissue .

• Consider sample preparation methods that might better preserve the KLB epitope.

Document all optimization steps methodically to establish a reliable protocol for future experiments.

How can I use KLB antibodies to investigate FGF21/FGFR signaling pathways?

KLB functions as a co-receptor that increases the ability of FGFR1 and FGFR4 to bind FGF21 , making KLB antibodies valuable tools for studying this signaling pathway:

• Co-immunoprecipitation Studies:

  • Use KLB antibodies to immunoprecipitate KLB protein complexes.

  • Probe for FGFR1, FGFR4, or FGF21 in the precipitate to assess direct interactions.

  • Compare interaction patterns in different metabolic states or disease models.

• Proximity Ligation Assays (PLA):

  • Combine KLB antibodies with antibodies against FGFR1/4 or FGF21.

  • PLA signals will reveal in situ protein-protein interactions at the single-molecule level.

  • Quantify interaction frequencies in different cell types or treatment conditions.

• Signaling Pathway Analysis:

  • Use KLB antibodies in conjunction with phospho-specific antibodies for downstream signaling molecules (ERK, AKT).

  • Compare signaling activation patterns in the presence/absence of FGF21 stimulation.

  • Block KLB using antibodies or inhibitors to assess the specificity of FGF21-mediated signaling effects.

• Tissue Distribution Studies:

  • Use immunohistochemistry with KLB antibodies to map receptor expression across tissues.

  • Correlate KLB expression with FGFR1/4 expression and FGF21 responsiveness.

  • Assess changes in receptor distribution in metabolic disease models.

This multi-faceted approach can provide comprehensive insights into how KLB mediates FGF21 signaling in various physiological and pathological contexts.

What methodologies can help resolve contradictory data when using different KLB antibodies?

Contradictory results with different KLB antibodies are not uncommon in research . To resolve such discrepancies:

• Epitope Mapping Analysis:

  • Determine the specific epitopes recognized by each antibody.

  • Consider whether these epitopes might be differentially affected by protein folding, post-translational modifications, or protein-protein interactions.

  • The sequence information provided for some KLB antibodies (e.g., amino acids 720-990 of human KLB ) can help in this assessment.

• Systematic Validation Approach:

  • Test multiple antibodies in parallel on the same samples.

  • Include appropriate positive and negative controls for each antibody.

  • Document specific experimental conditions where discrepancies occur.

• Complementary Methodologies:

  • Supplement antibody-based detection with non-antibody methods (e.g., mass spectrometry).

  • Use mRNA detection methods (qPCR, RNA-seq) to correlate with protein detection patterns.

  • Employ genetic approaches (siRNA, CRISPR) to validate specificity.

• Structured Contradiction Analysis:

  • Apply formal contradiction detection methodologies similar to those used in clinical literature .

  • Systematically document contradictory findings and the experimental conditions under which they occur.

  • Look for patterns that might explain discrepancies (e.g., tissue-specific isoforms, species differences).

• Literature Cross-Validation:

  • Compare your contradictory findings with published literature.

  • Contact antibody manufacturers for technical support and additional validation data.

  • Consider publishing contradictory findings to advance the field's understanding of KLB biology.

By employing these approaches, researchers can transform contradictory results into deeper insights about KLB biology and antibody behavior.

How can KLB antibodies be utilized in studying metabolic disorders?

KLB plays a critical role in metabolic regulation , making KLB antibodies valuable tools for investigating metabolic disorders:

• Expression Profiling in Disease Models:

  • Use Western blot and immunohistochemistry to compare KLB expression in healthy versus diseased tissues.

  • Quantify expression changes in key metabolic tissues (liver, adipose, pancreas) in response to diet, drugs, or genetic manipulations.

  • Correlate KLB expression with metabolic parameters (glucose tolerance, insulin sensitivity, lipid profiles).

• Mechanistic Studies:

  • Investigate KLB's role in cholesterol metabolism by studying its repression of CYP7A1 .

  • Examine KLB-mediated FGF21 signaling in various metabolic conditions.

  • Assess KLB's interaction with FGFRs in tissues implicated in metabolic disease.

• Therapeutic Target Validation:

  • Use KLB antibodies to monitor changes in expression or localization following experimental therapies.

  • Develop blocking antibodies against KLB to assess the effects of pathway inhibition.

  • Screen for compounds that modulate KLB expression or function.

• Biomarker Development:

  • Evaluate KLB as a potential biomarker for metabolic diseases.

  • Develop sensitive ELISA protocols using KLB antibodies for screening patient samples.

  • Correlate KLB levels with disease progression or therapeutic response.

• Multi-Omics Integration:

  • Combine KLB antibody-based proteomics with genomics, transcriptomics, and metabolomics data.

  • Create integrated models of KLB's role in metabolic regulation.

  • Identify novel interaction partners or signaling nodes in metabolic pathways.

These approaches can yield valuable insights into KLB's role in metabolic homeostasis and its potential as a therapeutic target in metabolic disorders.

What considerations are important for multiplex staining approaches involving KLB antibodies?

Multiplex staining allows simultaneous detection of multiple proteins in the same sample, providing valuable contextual information about KLB expression and function:

• Antibody Selection Criteria:

  • Choose KLB antibodies raised in different host species than other target antibodies to avoid cross-reactivity.

  • Verify that secondary antibodies do not cross-react with primary antibodies from different species.

  • Select KLB antibodies that work under compatible fixation and antigen retrieval conditions as other targets.

• Protocol Optimization:

  • Determine the optimal staining sequence—consider starting with the lowest abundance target.

  • Test whether sequential or simultaneous antibody incubation yields better results.

  • Optimize blocking steps to minimize background in multiplex contexts.

• Signal Separation Strategies:

  • Use differentially labeled secondary antibodies with non-overlapping emission spectra.

  • Consider tyramide signal amplification for weaker signals.

  • If using chromogenic detection, select enzymes and substrates that yield differently colored products.

• Controls for Multiplex Validation:

  • Include single-stain controls to confirm antibody specificity in the multiplex context.

  • Use blocking controls between sequential staining steps to prevent cross-reactivity.

  • Include tissue areas known to be positive or negative for each target as internal controls.

• Advanced Multiplex Technologies:

  • Consider cyclic immunofluorescence for detecting large numbers of targets sequentially.

  • Explore mass cytometry or imaging mass cytometry for high-parameter analysis.

  • Investigate proximity ligation assays for detecting KLB interactions with binding partners.

Careful validation and optimization of these parameters will enable successful multiplex staining approaches involving KLB antibodies, yielding rich contextual data about KLB biology.