KLF12 Antibody, FITC conjugated

What is KLF12 and what are its key biological functions?

KLF12 (Krueppel-like factor 12), also known as Transcriptional repressor AP-2rep, belongs to the Kruppel-like family of transcription factors that regulate multiple cellular processes including proliferation, differentiation, migration, and pluripotency . KLF12 contains zinc-finger domains crucial for DNA binding and has a Pro-Xaa-Asp-Leu-Ser (PVDLS) motif at the N-terminus that enables binding to the transcription corepressor C-terminal binding protein (CtBP) . It typically binds to the consensus sequence CAGTGGG in promoters of target genes . Research demonstrates KLF12 can function as either a tumor suppressor or inducer depending on signaling crosstalk and partners in specific cellular environments .

How does the FITC conjugation benefit detection methods for KLF12?

FITC (Fluorescein isothiocyanate) conjugation provides direct fluorescent visualization capabilities without requiring secondary antibody steps, streamlining immunofluorescence procedures. The 488nm excitation peak of FITC allows for detection using standard fluorescence microscopy and flow cytometry platforms. For KLF12 research, this conjugation is particularly valuable for cellular localization studies, as KLF12 has been shown to primarily localize in the nucleus where it interacts with p53 . The direct conjugation minimizes background and cross-reactivity issues that can occur with indirect detection methods, important when studying complex transcription factor networks like those involving KLF12.

What validation methods should be employed when first using KLF12 antibody?

Validation should include multiple complementary approaches. Begin with Western blot analysis using positive control lysates from cells known to express KLF12 (such as MCF-7 or ZR-75-30 breast cancer cells) . Expected molecular weight is approximately 45 kDa. Perform immunofluorescence staining to confirm nuclear localization pattern, as demonstrated in previous research where KLF12 co-localized with p53 in the nucleus . Include negative controls by testing in cells with KLF12 knockdown. For FITC-conjugated antibodies specifically, include an isotype control to account for non-specific binding. Consider performing cross-validation using an alternative KLF12 antibody from a different source to confirm specificity.

How should KLF12 antibody be incorporated in experiments studying p53-dependent transcriptional regulation?

When studying KLF12's role in p53-dependent transcriptional regulation, a multi-faceted experimental approach is essential. Begin with chromatin immunoprecipitation (ChIP) assays using the KLF12 antibody to identify KLF12-binding regions on target promoters like p21 . ChIP-Re-ChIP experiments would further confirm the co-occupancy of KLF12 and p53 on these promoters, as previous research has demonstrated both factors can be co-located on the p21 promoter . Complement this with luciferase reporter assays using p21-Luc and p53-Luc constructs to measure transcriptional activity with and without KLF12 overexpression or knockdown . Include p53 binding site mutations to distinguish between p53-dependent and p53-independent effects of KLF12 . For protein-protein interaction studies, co-immunoprecipitation experiments with KLF12 antibody can isolate p53-KLF12 complexes, which can be followed by Western blotting to detect associated proteins .

What controls are essential when using KLF12 antibody in cancer research?

In cancer research using KLF12 antibody, several critical controls must be implemented. Include cell lines with validated KLF12 knockdown (via siRNA or shRNA) and overexpression models as negative and positive controls respectively . When studying KLF12's effect on cancer cell proliferation, employ both MTT assays and colony formation assays to corroborate findings, as demonstrated in previous research where these complementary methods confirmed KLF12's proliferative effects . For flow cytometry applications, use isotype controls matched to the FITC-conjugated KLF12 antibody. When examining KLF12's impact on apoptosis pathways, include treatment controls such as cisplatin (CDDP) to induce genotoxic stress, as previous studies have shown KLF12 inhibits apoptosis in CDDP-treated cells . For transcriptional studies, include mutant binding site controls in reporter assays to confirm specificity .

How can multiplexed detection involving KLF12 be optimized?

Optimizing multiplexed detection with FITC-conjugated KLF12 antibody requires careful consideration of fluorophore combinations and spatial distribution of target proteins. Since KLF12 is FITC-labeled (green fluorescence), select compatible fluorophores for co-detection such as TRITC (red) or Cy5 (far-red) for other targets. When studying KLF12's interaction with p53, which predominantly occurs in the nucleus , include markers like DAPI for nuclear counterstaining. Use spectral imaging or sequential scanning to minimize bleed-through between channels. Consider implementing proximity ligation assays (PLA) to visualize and quantify KLF12-p53 interactions with spatial resolution. For flow cytometry applications, perform compensation controls and fluorescence-minus-one (FMO) controls to account for spectral overlap. When examining KLF12's relationship with the cell cycle, combine with propidium iodide staining for DNA content analysis, similar to previous cell cycle distribution experiments .

What are the optimal storage and handling procedures for maintaining FITC-conjugated KLF12 antibody activity?

FITC-conjugated KLF12 antibody requires specific storage and handling procedures to maintain fluorophore activity and antibody functionality. Store the antibody at -20°C or -80°C immediately upon receipt, as recommended by the manufacturer . Avoid repeated freeze-thaw cycles by aliquoting the antibody into single-use volumes before freezing. Protect from light during all handling steps, as FITC is susceptible to photobleaching. The antibody is supplied in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . When diluting for applications, use buffers with neutral pH (7.2-7.4) and consider adding 1% BSA to improve stability. For long-term storage of diluted antibody, add sodium azide (0.02%) as a preservative, but note that azide can interfere with some enzymatic detection systems. Document lot numbers, receipt dates, and freeze-thaw cycles to track antibody performance over time.

How should sample preparation be adjusted for optimal KLF12 detection using FITC-conjugated antibodies?

Sample preparation for optimal KLF12 detection requires careful consideration of fixation, permeabilization, and blocking steps. For cells expressing KLF12, particularly breast cancer cell lines like MCF-7 and ZR-75-30 , begin with fixation using 4% paraformaldehyde for 15 minutes at room temperature to preserve cellular architecture while maintaining protein antigenicity. Since KLF12 is primarily nuclear-localized and interacts with transcriptional machinery , thorough nuclear permeabilization is critical—use 0.25-0.5% Triton X-100 for 10 minutes. For blocking, employ 3-5% BSA in PBS for 30-60 minutes to reduce non-specific binding while preserving FITC fluorescence. When designing experiments to detect KLF12-p53 interactions, consider specialized fixation methods that better preserve protein-protein interactions, such as DSP (dithiobis(succinimidyl propionate)) crosslinking prior to standard fixation. For flow cytometry applications, gentle cell dissociation methods are preferred to maintain cellular integrity—use enzyme-free dissociation buffers when possible. When studying KLF12's relationship with cell cycle phases, synchronize cells using standard methods (serum starvation, double thymidine block) before antibody application.

What troubleshooting approaches address weak or non-specific KLF12 antibody signals?

When encountering weak or non-specific signals with FITC-conjugated KLF12 antibody, a systematic troubleshooting approach is necessary. For weak signals, first check antibody concentration—the KLF12 antibody is effective for ELISA applications , but optimal concentration may vary by application and cell type. Extend incubation time (overnight at 4°C rather than 1-2 hours at room temperature) to improve binding kinetics. For nuclear proteins like KLF12, ensure sufficient permeabilization by increasing Triton X-100 concentration to 0.5%. If non-specific binding occurs, implement more stringent washing steps (increase wash buffer volume and number of washes) and optimize blocking conditions (try 5% BSA with 0.1% Tween-20). Consider signal amplification methods such as tyramide signal amplification (TSA) if FITC signal is too weak. For flow cytometry applications showing high background, implement additional washing steps and adjust compensation settings. In Western blot applications, run positive controls from cells with known KLF12 expression levels (such as ZR-75-30 ) alongside experimental samples to better interpret signal specificity.

How can KLF12 antibody be utilized to investigate p53-dependent and p53-independent mechanisms?

Investigating KLF12's dual roles in p53-dependent and p53-independent mechanisms requires sophisticated experimental designs. For p53-dependent pathways, conduct co-immunoprecipitation experiments with KLF12 antibody followed by Western blotting for p53 to confirm their physical interaction, as previously demonstrated . Use ChIP-seq with KLF12 antibody to map genome-wide binding sites and compare with known p53 binding regions. To differentiate between mechanisms, implement reporter gene assays with wild-type p21 promoter constructs versus those with mutated p53 binding sites (p53-mut-p21-Luc), as previous research showed KLF12 can inhibit both constructs, indicating both p53-dependent and independent regulation . For detailed mechanism studies, examine the effect of KLF12 on p53 post-translational modifications using antibodies specific for acetylated lysine residues at positions 370/372/373, which have been shown to be critical for KLF12-mediated regulation of p53 stability . To investigate p53-independent functions, conduct experiments in p53-null cell lines with KLF12 overexpression or knockdown, focusing on direct KLF12 target genes identified through ChIP.

What methodologies effectively examine KLF12's role in modulating protein stability through post-translational modifications?

Examining KLF12's role in modulating protein stability through post-translational modifications requires specialized methodologies. To study KLF12's effect on p53 ubiquitination and acetylation , implement cycloheximide chase assays to measure protein half-life with and without KLF12 overexpression. Complementary in vitro ubiquitination assays with purified components can directly demonstrate KLF12's impact on ubiquitin ligase activity. For studying acetylation, perform immunoprecipitation with p53 antibodies followed by Western blotting with acetyl-lysine-specific antibodies, focusing on lysine residues 370/372/373 that have been shown to be critical for KLF12's effect on p53 . To investigate the mechanism of KLF12's disruption of p300-p53 interaction, conduct sequential immunoprecipitation experiments and proximity ligation assays. For quantitative assessment, implement SILAC (Stable Isotope Labeling with Amino acids in Cell culture) proteomics to measure changes in post-translational modification profiles under varying KLF12 expression levels. Use proteasome inhibitors like MG132 in parallel experiments to distinguish between effects on protein modification versus degradation, as previous research demonstrated MG132 treatment prevented KLF12-mediated reduction of p53 .

How can spatial transcriptomics be combined with KLF12 antibody detection to map transcriptional networks?

Combining spatial transcriptomics with KLF12 antibody detection offers powerful insights into transcriptional networks in complex tissues. Implement multiplexed immunofluorescence using FITC-conjugated KLF12 antibody alongside RNA in situ hybridization (RNA-ISH) for target genes like p21, whose transcription is directly regulated by KLF12 . For higher throughput applications, combine laser capture microdissection of KLF12-positive regions (identified using the antibody) with subsequent RNA-seq analysis. Newer technologies like Slide-seq or 10X Visium spatial transcriptomics can be coupled with KLF12 immunostaining on serial sections to correlate KLF12 protein localization with transcriptome-wide effects. For breast cancer tissue specifically, where KLF12 has demonstrated significant roles in proliferation and apoptosis resistance , develop tissue microarrays with tumor specimens at various stages and perform co-detection of KLF12 protein and key target transcripts. Analyze cellular heterogeneity by implementing single-cell approaches that combine protein detection (using index sorting with FITC-KLF12 antibody) and scRNA-seq to correlate KLF12 protein levels with transcriptional profiles at single-cell resolution.

How should researchers quantify and normalize KLF12 expression across different experimental conditions?

Quantification and normalization of KLF12 expression require rigorous statistical approaches tailored to the detection method. For immunofluorescence using FITC-conjugated KLF12 antibody, implement automated image analysis with nuclear segmentation to quantify nuclear intensity values, as KLF12 predominantly localizes to the nucleus . Normalize to nuclear area and include internal control regions within each image. For flow cytometry, report median fluorescence intensity (MFI) rather than percent positive cells, and normalize to isotype control values. When conducting Western blot analysis, use total protein normalization methods (such as stain-free technology) rather than single housekeeping genes, which may vary under experimental conditions affecting transcription factors. For experiments comparing KLF12 expression across multiple cell lines or treatment conditions, implement a standard curve using recombinant KLF12 protein at known concentrations. In qPCR studies measuring KLF12 mRNA levels, carefully select reference genes that remain stable under experimental conditions affecting transcription factors—validate multiple reference genes using algorithms like geNorm or NormFinder. For all quantification methods, report biological replicates (n≥3) and appropriate statistical tests with exact p-values.

How can researchers distinguish between direct and indirect effects of KLF12 on gene expression?

Distinguishing between direct and indirect effects of KLF12 on gene expression requires a multifaceted approach. First, conduct ChIP-seq with KLF12 antibody to identify genome-wide binding sites, focusing on regions containing the consensus KLF12 binding sequence (CAGTGGG) . For candidate genes like p21, perform motif analysis to identify potential KLF12 binding sites, followed by site-directed mutagenesis of these sites in reporter constructs to confirm functional relevance, as demonstrated in previous research where mutation of KLF12-binding sites reduced its inhibitory effect on p21 transcription . Implement time-course experiments after inducible KLF12 expression to differentiate between immediate (likely direct) and delayed (possibly indirect) transcriptional responses. For suspected indirect mechanisms involving other transcription factors (like p53), conduct epistasis experiments with knockdown or knockout of the intermediate factor. Perform RNA-seq after KLF12 perturbation under conditions where protein synthesis is inhibited (using cycloheximide) to isolate direct transcriptional effects. Combine these approaches with metabolic labeling of newly synthesized RNA (such as 4sU-seq) to capture immediate transcriptional changes following KLF12 modulation.

How should contradictory findings regarding KLF12's role as tumor suppressor versus oncogene be reconciled?

Reconciling contradictory findings regarding KLF12's dual roles as tumor suppressor or oncogene requires careful consideration of biological context. Previous research has indicated that KLF12 can function as either a tumor suppressor or inducer depending on signaling crosstalk and partners in specific cellular environments . When analyzing seemingly contradictory data, first examine cell type specificity—KLF12 promotes proliferation in breast cancer cells like MCF-7 and ZR-75-30 , but may have different effects in other tissues. Consider the influence of experimental systems—in vitro versus in vivo studies may yield different results due to microenvironmental factors affecting KLF12 function. Analyze protein interaction networks in each model system, as KLF12's effect may depend on the presence of specific binding partners like p53 or p300 . Examine post-translational modifications of KLF12 itself, which may switch its function from activator to repressor. Investigate potential isoform-specific effects by confirming which KLF12 variants are being studied. For clinical correlations, stratify patient cohorts based on molecular subtypes and disease stages—for example, microRNA-205 targeting of KLF12 has been reported specifically in basal-like breast carcinoma . Finally, implement systems biology approaches to model the dynamic behavior of KLF12 within gene regulatory networks under varying conditions.