Krt18 Antibody, Biotin conjugated

What is Cytokeratin 18 (KRT18) and where is it expressed in tissues?

Cytokeratin 18 (KRT18) belongs to the type A (acidic) subfamily of low molecular weight keratins and typically exists in combination with cytokeratin 8. KRT18 is widely expressed in simple epithelia throughout the body but notably absent in stratified squamous epithelia. Specific tissue distribution includes the gastrointestinal tract, respiratory tract, urogenital tract, as well as endocrine and exocrine tissues and mesothelial cells . KRT18 is also found in epithelial tumors of various origins including gastrointestinal tract, lung, breast, pancreas, ovary, and thyroid . Importantly, while tissues from the gastrointestinal tract are positive for both cytokeratin 8 and 18, they typically do not contain cytokeratin 14, which provides a useful differentiation marker .

What are the functional roles of KRT18 in cellular processes?

KRT18 serves several critical functions in cellular biology beyond its structural role. It is involved in the uptake of thrombin-antithrombin complexes by hepatic cells and plays a key role in filament reorganization when phosphorylated . Additionally, KRT18 participates in the delivery of mutated CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) to the plasma membrane. Together with KRT8, it is also involved in interleukin-6 (IL-6)-mediated barrier protection, highlighting its importance in immune response regulation . These diverse functions make KRT18 an important target for research in multiple fields including hepatology, oncology, and immunology.

What applications are biotin-conjugated KRT18 antibodies suitable for?

Biotin-conjugated KRT18 antibodies are versatile research tools applicable across multiple experimental platforms. They are primarily validated for Western Blot (WB) applications with human samples, as demonstrated by their successful use with cell lines such as HT-29 (human colorectal adenocarcinoma) and MOLT-4 (human lymphoblastic leukemia) . While Western blotting represents the primary validated application, the literature suggests these antibodies may potentially be used in other biotin-streptavidin detection systems including immunohistochemistry and immunoprecipitation, though researchers should perform validation studies for these specific applications . The biotin conjugation enables signal amplification through secondary detection with streptavidin-conjugated reporters, which can be particularly valuable for detecting low abundance targets.

How should sample preparation conditions be optimized for KRT18 detection in Western blotting?

Sample preparation significantly impacts the quality of KRT18 detection in Western blotting applications. Both reducing and non-reducing conditions have been successfully employed, though different patterns may emerge. For example, Western blot analysis using biotin-conjugated anti-Cytokeratin 18 antibody [C-04] (ab27553) at 2 μg/mL concentration revealed variations in band patterns between reducing and non-reducing conditions . When using HT-29 whole cell lysates under reducing conditions, researchers observed the expected KRT18 band pattern, while MOLT-4 samples showed different results under non-reducing conditions . Additionally, researchers should be aware that proteolytic fragments of approximately 25 kDa may be observed in certain sample preparations . Optimal lysis buffers typically contain protease inhibitors to prevent degradation of KRT18 during sample processing.

What are the recommended antibody dilutions and incubation conditions for different applications?

Optimal antibody dilutions vary significantly based on the specific application and detection method. For Western blot applications, biotin-conjugated KRT18 antibodies are typically used at concentrations around 2 μg/mL . For other KRT18 antibody formats, recommended dilutions include 1:2000-1:10000 for Western blot, 1:50-1:500 for immunohistochemistry, and 1:50-1:500 for immunofluorescence/ICC applications . These ranges provide starting points, but researchers should optimize concentrations for their specific experimental systems and detection methods. Incubation conditions typically involve overnight incubation at 4°C for primary antibodies, though this should be empirically determined based on the specific antibody clone and application.

How can researchers troubleshoot non-specific binding with biotin-conjugated KRT18 antibodies?

Non-specific binding is a common challenge when working with biotin-conjugated antibodies. Several strategies can minimize this issue when working with KRT18 antibodies. First, researchers should implement appropriate blocking steps using BSA or normal serum from the same species as the secondary detection reagent. Second, if working with tissues or cells that may contain endogenous biotin, consider using avidin/biotin blocking kits prior to antibody application. Third, optimize antibody concentration through titration experiments, as excessive antibody concentrations often lead to increased background. For fluorescence applications, note that blue fluorescent dyes like CF®405S may give higher non-specific background than other dye colors and are not recommended for detecting low abundance targets . Finally, including proper negative controls (isotype controls, secondary-only controls, and samples known to be negative for KRT18) helps distinguish specific from non-specific signals.

What positive and negative controls should be included in KRT18 antibody experiments?

Proper control selection is crucial for interpreting KRT18 antibody experimental results. For positive controls, cell lines with documented KRT18 expression should be included. HT-29 (human colorectal adenocarcinoma), A431, A549, HCT 116, and HepG2 cells have been validated as positive for KRT18 expression and can serve as reliable positive controls . For tissue samples, liver tissue from human, mouse, or rat sources provides consistent KRT18 positivity . Negative controls should include MOLT-4 cells for Western blot applications, as these lymphoblastic leukemia cells show minimal KRT18 expression . For technical controls, include an isotype control (same immunoglobulin class and conjugate as the KRT18 antibody) and secondary-only controls to assess non-specific binding. When working with multiple species, ensure that the selected KRT18 antibody has been validated for cross-reactivity with your target species, as some antibodies may show different affinity across human, mouse, and rat samples .

How does sample fixation impact KRT18 epitope accessibility and detection sensitivity?

Sample fixation methodology significantly impacts epitope accessibility and detection outcomes for KRT18. For immunohistochemical applications, antigen retrieval is typically required, with two main approaches yielding success: TE buffer at pH 9.0 or citrate buffer at pH 6.0 . The higher pH TE buffer often yields superior results for KRT18 detection, particularly in formalin-fixed paraffin-embedded (FFPE) tissues where cross-linking can mask epitopes. For immunofluorescence applications on cultured cells, 4% paraformaldehyde fixation for 15-20 minutes followed by permeabilization with 0.1-0.5% Triton X-100 generally preserves KRT18 structure while allowing antibody access. Methanol fixation can provide an alternative approach that simultaneously fixes and permeabilizes cells, but may alter certain epitopes. Researchers should compare multiple fixation protocols when establishing a new assay, as KRT18 detection sensitivity can vary dramatically based on these parameters.

What factors should guide selection between different KRT18 antibody clones?

Several factors should inform the selection between different KRT18 antibody clones. First, consider the specific epitope recognized by each clone and whether that region may be masked or modified in your experimental system. The C-04 clone and DA-7 clone offered as biotin conjugates have different epitope specificities, which may impact their performance in different applications . Second, evaluate validation data for your specific application - some clones perform better in Western blotting while others excel in immunohistochemistry or immunofluorescence. Third, consider species cross-reactivity if working with non-human samples; many KRT18 antibodies are validated for human, mouse, and rat samples, but performance may vary . Finally, assess the literature using specific clones for your application of interest, as published studies can provide valuable insights into antibody performance in specific experimental contexts.

How should researchers interpret proteolytic fragments of KRT18 in Western blotting?

Proteolytic fragments of KRT18 observed in Western blotting require careful interpretation as they may represent biologically significant events rather than technical artifacts. A proteolytic fragment of approximately 25 kDa has been documented with certain KRT18 antibodies . This fragment may result from caspase-mediated cleavage during apoptosis, as KRT18 is a known caspase substrate. When analyzing such fragments, researchers should first verify they are not artifacts of sample preparation by including protease inhibitors in lysis buffers. Next, compare the pattern of fragmentation across different experimental conditions, as changes in the relative abundance of full-length versus fragmented KRT18 may indicate altered cellular processes. Finally, consider complementary approaches like using antibodies specific to caspase-cleaved KRT18 to confirm the identity of these fragments. In cancer research particularly, these fragments may serve as biomarkers for tumor cell death and treatment response.

How can quantitative comparisons of KRT18 expression be standardized across different experimental conditions?

Standardizing quantitative comparisons of KRT18 expression requires rigorous methodological approaches. For Western blot analysis, normalization to appropriate loading controls is essential - β-actin works for most applications, though GAPDH may be preferred in certain contexts. Densitometric analysis should include multiple biological replicates (minimum n=3) with technical replicates to ensure statistical validity. For immunohistochemical quantification, establish consistent scoring methodologies that account for both staining intensity and percentage of positive cells, potentially using the H-score or Allred scoring systems. Digital image analysis with consistent thresholding parameters can reduce subjective bias in quantification. When comparing KRT18 expression across different tissue or cell types, remember that baseline expression levels vary naturally, so fold-change relative to appropriate control samples for each tissue/cell type provides more meaningful data than absolute expression levels. Finally, ensure that antibody concentrations and detection systems remain consistent across all samples being compared.

What approaches help distinguish between specific KRT18 signal and background in complex tissue samples?

Distinguishing specific KRT18 signal from background in complex tissue samples requires multiple technical approaches. First, implement proper blocking strategies using either 5% BSA or 5-10% normal serum matched to the secondary antibody species. Second, titrate primary antibody concentration to determine the optimal signal-to-noise ratio; biotin-conjugated KRT18 antibodies typically perform well at 2 μg/mL for Western blot applications, but this should be optimized for each tissue type . Third, include anatomical positive and negative controls within each experiment - KRT18 is absent in stratified squamous epithelia, making these regions effective negative controls, while simple epithelia provide positive controls . Fourth, for fluorescence applications, be aware that certain fluorophores like CF®405S may generate higher background and are not recommended for low abundance targets . Finally, parallel staining with multiple KRT18 antibody clones recognizing different epitopes can confirm signal specificity, as genuine KRT18 expression should be detected by multiple antibodies while non-specific binding patterns typically differ between antibody clones.

What are the optimal approaches for multiplex immunofluorescence including KRT18 detection?

Multiplex immunofluorescence incorporating KRT18 detection requires careful planning of antibody combinations and detection strategies. When using biotin-conjugated KRT18 antibodies, pair them with streptavidin conjugated to a fluorophore with minimal spectral overlap with other channels. For optimal fluorophore selection with KRT18 antibodies, researchers have multiple options with distinct excitation/emission profiles: CF®488A (490/515nm, GFP/FITC channel), CF®568 (562/583nm, RFP/TRITC channel), CF®594 (593/614nm, Texas Red channel), or CF®640R (642/662nm, far-red channel) . The choice depends on other markers in your multiplex panel and available microscopy filter sets. For co-staining with KRT8 (which often co-expresses with KRT18), use antibodies raised in different host species or directly conjugated to different fluorophores to avoid cross-reactivity. Sequential staining protocols rather than cocktail approaches may be necessary for complex multiplex panels to minimize cross-reactivity. Finally, include single-color controls for each fluorophore to establish proper compensation settings during image acquisition and analysis.

What dilution series should be tested when optimizing biotin-conjugated KRT18 antibodies for Western blotting?

When optimizing biotin-conjugated KRT18 antibodies for Western blotting, a systematic dilution series should be tested to determine optimal concentration. Based on published protocols, begin with 2 μg/mL as a starting point, which has proven effective with both the C-04 and DA-7 clones on human cell line samples . From this reference point, test a 2-fold dilution series ranging from 0.5 μg/mL to 4 μg/mL to identify the concentration providing the best signal-to-noise ratio. For each dilution, use consistent sample types (e.g., HT-29 cell lysate as a positive control) and detection methods. When working with streptavidin-HRP as a secondary detection reagent, a similar titration approach should be employed, typically testing dilutions between 1:1000 and 1:10000. Load consistent amounts of protein (typically 20-30 μg total protein per lane) across all test conditions. Visualization methods (chemiluminescence, fluorescence) may also affect optimal antibody concentration, so the final optimization should be performed using your intended detection system.

How should researchers approach troubleshooting weak or absent KRT18 signal in Western blotting?

When troubleshooting weak or absent KRT18 signal in Western blotting, researchers should systematically evaluate each step of the protocol. First, verify sample integrity by testing for a housekeeping protein like β-actin or GAPDH using the same membrane. Second, confirm that the sample type is appropriate - HT-29, A431, A549, HCT 116, and HepG2 cells are reliable positive controls for KRT18 expression . Third, examine sample preparation conditions - KRT18 detection can differ between reducing and non-reducing conditions, with some antibody clones performing better under specific conditions . Fourth, consider increasing protein loading to 40-50 μg per lane if KRT18 is expressed at low levels in your samples. Fifth, extend primary antibody incubation time to overnight at 4°C if standard protocols yield weak signals. Sixth, switch to a more sensitive detection system, such as enhanced chemiluminescence (ECL) substrate. Finally, try alternative membrane blocking reagents, as some blocking solutions may mask the epitope recognized by your specific antibody clone.

How can KRT18 antibodies be used in circulating tumor cell (CTC) research?

KRT18 antibodies have significant applications in circulating tumor cell (CTC) research due to the epithelial origin of many cancer types. For CTC detection protocols, biotin-conjugated KRT18 antibodies can be incorporated into immunomagnetic separation systems or flow cytometry panels. When designing such assays, researchers should consider several technical factors. First, use KRT18 antibodies in combination with other epithelial markers like EpCAM to improve specificity, as KRT18 is expressed across multiple epithelial tumors including those from gastrointestinal tract, lung, breast, pancreas, ovary, and thyroid origins . Second, incorporate CD45 antibodies as a negative selection marker to exclude leukocytes, which are KRT18-negative. Third, when working with blood samples, optimize red blood cell lysis protocols to minimize background without compromising CTC viability or marker expression. Fourth, consider the fragility of CTCs and the potential for KRT18 degradation during sample processing by including protease inhibitors and processing samples rapidly. Future directions in this field include developing multiplexed approaches that simultaneously assess KRT18 expression alongside tumor-specific markers and genetic alterations.

What is the role of KRT18 in distinguishing between different cancer subtypes?

KRT18 expression patterns can help distinguish between different cancer subtypes, making KRT18 antibodies valuable diagnostic tools. KRT18 is consistently expressed in adenocarcinomas arising from simple epithelia but is typically absent in squamous cell carcinomas, reflecting the normal tissue distribution where KRT18 is found in simple epithelia but not stratified squamous epithelia . When designing panels for tumor classification, combine KRT18 with other cytokeratin markers - for example, KRT7/KRT20 profiling alongside KRT18 can help distinguish between colorectal, ovarian, and breast adenocarcinomas. For hepatocellular carcinoma research, KRT18 is particularly valuable as it is expressed in hepatocytes and can help distinguish primary liver tumors from metastases. Quantitative assessment of KRT18 expression levels, rather than simple presence/absence, may provide prognostic information in certain cancer types. Researchers should note that during epithelial-mesenchymal transition (EMT), cancer cells may downregulate KRT18 expression, which can complicate interpretation in metastatic settings. Future research directions include correlating KRT18 expression patterns with molecular subtypes defined by genomic and transcriptomic profiling.

How can phosphorylation status of KRT18 be assessed using biotin-conjugated antibodies?

Assessing KRT18 phosphorylation status requires specialized approaches that can be adapted for use with biotin-conjugated antibodies. KRT18 phosphorylation plays a key role in filament reorganization and cellular responses to stress, making it an important regulatory mechanism to study . To investigate phosphorylation states, researchers can employ a two-step approach: first use phosphorylation-specific KRT18 antibodies (targeting specific phosphorylation sites like Ser33) followed by detection with biotin-conjugated pan-KRT18 antibodies to assess the ratio of phosphorylated to total KRT18. Alternatively, researchers can perform immunoprecipitation with biotin-conjugated KRT18 antibodies, followed by Western blotting with phospho-specific antibodies. When analyzing phosphorylation patterns, include appropriate controls such as phosphatase-treated samples (negative control) and samples treated with phosphatase inhibitors (positive control). Stimulation with EGF or treatment with okadaic acid can increase KRT18 phosphorylation levels, providing useful positive controls. For quantitative assessment, consider using Phos-tag™ acrylamide gels, which can separate phosphorylated from non-phosphorylated KRT18 based on mobility shifts, followed by detection with biotin-conjugated KRT18 antibodies.