KLF13 Antibody

What is KLF13 and what are its key biological functions?

KLF13 (Krüppel-like factor 13) is a transcription factor belonging to the KLF family, characterized by three classical zinc finger DNA-binding domains with a C2H2 motif structure. These zinc finger domains are tetrahedrally coordinated by 2 cysteines and 2 histidines around a zinc atom . KLF13 functions primarily by binding to CG-rich sequences, GT boxes, and CACCC boxes within promoter regions of target genes .

Functionally, KLF13 plays crucial roles in:

• Transcriptional activation of genes in erythroid lineage cells, including GATA1 and glycophorin B

• Binding to the A and A/B RANTES promoter regions, affecting immune cell function

• Cell proliferation and differentiation pathways

• Metabolic regulation, with clinical studies showing its involvement in obesity

The multifunctional nature of this transcription factor makes KLF13 antibodies essential tools for investigating diverse biological processes.

What applications are KLF13 antibodies validated for?

KLF13 antibodies have been validated for multiple research applications based on comprehensive testing. The table below summarizes the validated applications from analyzed sources:

For Western blot applications, KLF13 antibodies have been successfully tested on multiple sample types including human cell lines (HT-29 cells), mouse cell lines (L929 cells), and rat liver tissue . For immunohistochemistry, positive detection has been confirmed in human brain tissue, with recommended antigen retrieval using TE buffer at pH 9.0 or alternatively with citrate buffer at pH 6.0 .

Many KLF13 antibodies have been cited in peer-reviewed research, with one product showing 12 citations for Western blot usage, 5 for IHC, and 3 for IF applications , demonstrating their reliability in various experimental contexts.

What species reactivity do commercial KLF13 antibodies demonstrate?

The species reactivity of KLF13 antibodies varies by product but generally includes main model organisms used in research. Based on the analyzed data:

When selecting a KLF13 antibody for cross-species applications, researchers should consider the degree of sequence homology between their target species and the immunogen used to generate the antibody. The immunogen information is particularly important—for example, antibodies raised against the epitope corresponding to positions 74-169 of mouse KLF13 may recognize KLF13 in other vertebrates due to sequence conservation in this region .

What is the expected molecular weight of KLF13 protein in experimental detection?

An important consideration when validating KLF13 antibody performance is understanding the expected molecular weight pattern. The search results reveal an interesting discrepancy between calculated and observed molecular weights:

This difference between calculated (31 kDa) and observed (37-45 kDa) molecular weights is significant for experimental interpretation. The higher observed molecular weight likely results from post-translational modifications such as phosphorylation, glycosylation, or SUMOylation that affect protein migration in SDS-PAGE gels .

What are the optimal storage conditions for KLF13 antibodies?

Proper storage conditions are critical for maintaining antibody functionality and experimental reproducibility. Based on manufacturer recommendations:

For most KLF13 antibodies, storage at -20°C is sufficient, though some products require -80°C storage . The presence of glycerol in the storage buffer acts as a cryoprotectant, preventing damage during freeze-thaw cycles.

How can I optimize Western blot protocols for KLF13 detection?

Optimizing Western blot protocols for KLF13 detection requires attention to several key methodological factors:

Sample Preparation Considerations:

• Use fresh tissue/cell lysates when possible, as KLF13 may be subject to proteolytic degradation

• Include phosphatase inhibitors in lysis buffers to preserve post-translational modifications, as KLF13 shows a higher observed molecular weight (37-45 kDa) than calculated (31 kDa)

• Load adequate protein (30-50 μg total protein per lane) to ensure detection of potentially low-abundance transcription factors

Blocking and Antibody Incubation:

• For polyclonal KLF13 antibodies, use 5% non-fat milk or BSA in TBST for blocking

• Follow manufacturer-recommended dilutions (typically 1:1000-1:4000 for Western blot)

• Consider overnight incubation at 4°C for primary antibody to maximize specific binding

• Use validated secondary antibodies such as goat anti-rabbit IgG conjugated with HRP for detection

The Western blot validation data for KLF13 antibodies shows successful detection in cell lines such as L929 and HT-29, as well as in rat liver tissue . When troubleshooting weak signals, consider extending primary antibody incubation time or using enhanced chemiluminescence substrates with higher sensitivity.

What are the critical considerations for immunohistochemistry with KLF13 antibodies?

Successful immunohistochemistry (IHC) with KLF13 antibodies requires careful attention to tissue processing and antigen retrieval methods:

Antigen Retrieval Optimization:

• Primary recommendation: TE buffer at pH 9.0

• Alternative method: Citrate buffer at pH 6.0

The choice between these two methods can significantly impact staining quality. TE buffer at higher pH (9.0) often provides better unmasking of epitopes in formalin-fixed tissues for nuclear antigens like transcription factors.

Antibody Dilution Range:

• Start with a dilution range of 1:20-1:200 for IHC applications

• Perform titration experiments to determine optimal concentration for your specific tissue type

Positive Control Selection:

• Human brain tissue has been validated as a positive control for KLF13 IHC

• Consider including multiple control tissues in method development

For fluorescent detection in IF applications, KLF13's nuclear localization should be confirmed by co-staining with nuclear markers such as DAPI. Researchers should expect nuclear staining pattern consistent with KLF13's function as a transcription factor, though some cytoplasmic staining may be observed during certain cellular states.

How can I validate KLF13 antibody specificity in my experimental system?

Rigorous validation of antibody specificity is crucial for generating reliable research data. For KLF13 antibodies, consider implementing these approaches:

Genetic Validation:

• Knockdown/knockout validation: Use siRNA, shRNA, or CRISPR/Cas9 to deplete KLF13 and confirm reduction/absence of signal

• The search results indicate multiple published studies using KLF13 knockdown/knockout validation (3 cited publications)

Peptide Competition Assays:

• Pre-incubate antibody with immunizing peptide before application

• For antibodies using synthetic peptide immunogens near K166 or the 74-169 aa region, obtain corresponding blocking peptides

Cross-validation Methods:

• Compare results from multiple KLF13 antibodies targeting different epitopes

• Use antibodies raised against different regions (N-terminal vs. C-terminal) to confirm consistent detection patterns

Recombinant Expression:

• Overexpress tagged KLF13 in a cellular system and confirm detection at the expected molecular weight range (37-45 kDa)

The observed molecular weight discrepancy (calculated 31 kDa vs. observed 37-45 kDa) should be considered when interpreting validation results, as it indicates potential post-translational modifications that may affect epitope accessibility in different experimental contexts .

How do post-translational modifications affect KLF13 antibody detection?

Post-translational modifications (PTMs) of KLF13 present important considerations for antibody-based detection:

Evidence of Post-translational Modifications:

• The difference between calculated (31 kDa) and observed (37-45 kDa) molecular weights strongly suggests the presence of PTMs

• KLF13 contains a non-acetylation site at K166, which is specifically referenced in antibody design

Impact on Epitope Accessibility:

• Antibodies designed against regions near PTM sites may show differential binding depending on the modification state

• The antibody from Antibodies.com (A40572) is specifically designed around the non-acetylation site of K166, suggesting acetylation may occur at this residue under certain conditions

Experimental Considerations:

• When studying KLF13 in contexts where PTM status may change (e.g., cell signaling studies, differentiation models), consider using antibodies targeting different epitopes

• For phosphorylation studies, include phosphatase inhibitors in sample preparation

• For acetylation studies, consider HDAC inhibitor treatment to preserve acetylation states

The potential for multiple PTMs explains the observed molecular weight range (37-45 kDa) and suggests researchers may observe multiple bands or band shifts depending on the experimental context and cell type being studied .

What experimental approaches are recommended for studying KLF13's role in transcriptional regulation?

As a transcription factor, KLF13 regulates gene expression through binding to specific DNA sequences. To study its transcriptional regulatory function:

Chromatin Immunoprecipitation (ChIP) Approaches:

• Select KLF13 antibodies validated for immunoprecipitation applications

• Design primers targeting predicted binding regions containing CG-rich sequences, GT boxes, or CACCC boxes

• Include positive controls such as known KLF13 targets (GATA1, glycophorin B, RANTES promoter regions)

Reporter Gene Assays:

• Construct reporters containing KLF13 binding sites (CG-rich sequences, GT and CACCC boxes)

• Perform co-transfection experiments with KLF13 expression constructs

• Use KLF13 antibodies to confirm expression levels by Western blot in parallel

Gene Expression Analysis:

• Combine KLF13 overexpression or knockdown with RNA-seq or qPCR of target genes

• Validate changes in protein levels of targets using appropriate antibodies

• Correlate with immunohistochemistry data in relevant tissues

When designing these experiments, consider KLF13's known roles in specific biological contexts, such as erythroid lineage gene regulation (GATA1, glycophorin B) or its involvement in obesity-related pathways . The choice of appropriate cell lines or tissue models should reflect these specific regulatory functions.

What are the experimental considerations when studying KLF13 in different disease models?

KLF13 has been implicated in various disease processes, requiring specific experimental considerations when studied in disease models:

Obesity Research:

• KLF13 has a clinically established role in obesity

• Consider adipose tissue-specific expression analysis

• Compare KLF13 expression levels between obese and non-obese models using validated antibodies for Western blot and IHC

• Design experiments to investigate metabolic pathway interactions

Cancer Research:

• KLF13 is involved in cell proliferation and differentiation pathways relevant to cancer

• Compare expression in tumor vs. normal tissue using IHC with appropriate controls

• Analyze correlation between KLF13 expression levels and clinical outcomes

• Consider cell line models representing different cancer stages

Immune System Studies:

• Given KLF13's role in binding RANTES promoter regions , design experiments investigating:

  • T-cell activation and cytokine production

  • Inflammatory responses in various disease models

  • Correlation with other immune regulatory factors

For all disease models, researchers should validate antibody performance in the specific tissue or cell type being studied, as expression levels and post-translational modifications may vary significantly between different pathological conditions. The reactivity of KLF13 antibodies with human, mouse, and rat samples enables translational research spanning from animal models to human clinical samples.

How should I select between different KLF13 antibodies for specific research questions?

Selecting the optimal KLF13 antibody requires matching antibody characteristics to your specific experimental requirements:

Immunogen Considerations:

• For studies focused on specific domains or modifications, select antibodies raised against relevant regions:

  • Antibodies.com (A40572): Targets region around non-acetylation site K166

  • Kerafast antibodies: Target epitope corresponding to positions 74-169

• For general KLF13 detection, consider antibodies raised against full fusion proteins

Application-Specific Selection:

• For Western blot: All analyzed antibodies are validated for WB

• For immunohistochemistry: Select antibodies with published IHC validation

• For immunofluorescence: Verify IF validation in similar tissue/cell types to your model

Cross-reactivity Requirements:

• For multi-species studies: Select antibodies with demonstrated reactivity across target species

• For human-specific studies: Consider human-optimized products

The combination of immunogen information, validated applications, and species reactivity should guide selection based on your specific experimental requirements and biological system.

What controls should I include when using KLF13 antibodies in my experiments?

Proper experimental controls are essential for generating reliable and interpretable data with KLF13 antibodies:

Positive Controls:

• Cell/Tissue Types:

  • HT-29 cells and L929 cells for Western blot

  • Human brain tissue for IHC

  • Rat liver tissue for Western blot

• Recombinant Expression: Consider tagged KLF13 expression as a positive control

Negative Controls:

• Genetic: KLF13 knockdown or knockout samples when available

• Technical:

  • Primary antibody omission

  • Isotype control antibody (rabbit IgG at equivalent concentration)

  • Secondary antibody-only controls

Specificity Controls:

• Peptide competition assays using immunizing peptides

• Multiple antibody validation using different KLF13 antibodies targeting distinct epitopes

Including these controls allows for confident interpretation of results and troubleshooting of any technical issues that may arise. Particularly for nuclear transcription factors like KLF13, proper controls help distinguish specific nuclear staining from background or non-specific signals.

What are the recommended approaches for multiplexing KLF13 detection with other markers?

Multiplexing KLF13 detection with other markers is valuable for characterizing its role in complex biological contexts:

Co-immunofluorescence Approaches:

• Select KLF13 antibodies raised in rabbit and pair with antibodies raised in different host species (mouse, goat, etc.) for other markers

• Use secondary antibodies with non-overlapping fluorophore spectra

• Include single-color controls to assess bleed-through

• Consider spectral unmixing for closely overlapping fluorophores

Sequential Immunohistochemistry:

• For multiple rabbit antibodies, consider sequential detection protocols with complete stripping or blocking between rounds

• Validate stripping efficiency by confirming absence of signal after primary antibody removal

• Document tissue integrity after multiple staining rounds

Marker Selection Strategies:

• Pair KLF13 with relevant transcription factors in the same regulatory pathways

• For obesity studies, consider adipocyte markers

• For cell differentiation studies, combine with lineage-specific markers

• For transcriptional regulation studies, co-stain with RNA polymerase II to assess active transcription sites

When planning multiplexing experiments, the nuclear localization of KLF13 provides a distinct compartmentalization that facilitates co-localization analysis with cytoplasmic or membrane markers.