MYLK4 Antibody

What is MYLK4 and what are its key biological functions?

MYLK4 (Myosin Light Chain Kinase Family Member 4) is also known as SGK085 (Sugen kinase 85) and belongs to the CAMK Ser/Thr protein kinase family . It functions as a calcium/calmodulin-dependent enzyme, requiring Ca²⁺-calmodulin complexes for activation along with Mg²⁺ or Ca²⁺ as co-factors . MYLK4 exists in two isoforms produced by alternative splicing, with a calculated molecular weight of 388 amino acids (45 kDa) . As part of the myosin light chain kinase family, MYLK4 likely plays roles in muscle contraction, cell motility, and cytoskeletal organization, though specific functions may vary across tissue types. The protein has been detected in various human tissues, with positive detection noted particularly in heart tissue and tonsillitis tissue in immunohistochemical analyses .

What applications are MYLK4 antibodies typically used for?

MYLK4 antibodies are validated for multiple experimental applications based on the search results. The most common applications include:

For optimal results in immunohistochemistry applications, antigen retrieval is typically suggested with TE buffer pH 9.0, with citrate buffer pH 6.0 as an alternative option .

What are the common storage and handling recommendations for MYLK4 antibodies?

Most MYLK4 antibodies require specific storage conditions to maintain their efficacy and stability. Standard recommendations include storage at -20°C, where they remain stable for approximately one year after shipment . The antibodies are typically supplied in liquid form, often in PBS with additives such as 50% glycerol, 0.02% sodium azide, and sometimes 0.5% BSA to enhance stability . According to manufacturer guidelines, some formulations specify that aliquoting is unnecessary for -20°C storage, though this may vary by product . For small volume formats (e.g., 20μl), some preparations contain 0.1% BSA to prevent protein loss due to binding to tube walls . Repeated freeze-thaw cycles should be avoided to maintain antibody performance and integrity of the protein structure .

How should researchers optimize MYLK4 antibody dilutions for different applications?

Optimizing antibody dilutions is critical for achieving specific signal with minimal background. For MYLK4 antibodies, recommended dilutions vary significantly by application:

For Western Blot applications, a wide dilution range of 1:500-1:5000 is typically recommended, with many manufacturers suggesting starting points around 1:1000-1:4000 . The optimal dilution should be determined experimentally for each specific cell or tissue type. For example, when using the antibody with HEK-293 or L02 cells, starting with a 1:1000 dilution and then refining based on signal strength and background is advisable .

For Immunoprecipitation, the recommended amount is typically 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate . This application requires careful optimization of antibody-to-lysate ratio to achieve efficient pull-down without non-specific binding.

For Immunohistochemistry, dilutions between 1:20-1:400 are recommended . Researchers should conduct preliminary experiments with a dilution series on control tissues (such as human heart or tonsillitis tissue, which have shown positive results) to determine optimal conditions. The antigen retrieval method significantly impacts results, with TE buffer pH 9.0 generally preferred, though citrate buffer pH 6.0 can serve as an alternative .

For ELISA applications, much higher dilutions (1:20000-1:80000) may be appropriate due to the high sensitivity of this method . Researchers should titrate the antibody in each specific testing system to obtain optimal results, particularly when working with different sample types or detection systems .

What validation methods should be used to confirm MYLK4 antibody specificity?

Validating antibody specificity is essential for ensuring reliable experimental results. For MYLK4 antibodies, several complementary validation approaches are recommended:

• Knockout/Knockdown Controls: Utilizing MYLK4 knockout or knockdown samples provides the most stringent specificity validation. Published studies have employed this method, demonstrating the utility of MYLK4 antibodies in KD/KO experimental systems .

• Western Blot Analysis: Confirming the detection of a single band at the expected molecular weight (45 kDa for MYLK4) in positive control samples such as HEK-293 or L02 cells . Any additional bands should be investigated as potential isoforms, degradation products, or non-specific binding.

• Immunoprecipitation Validation: Using IP followed by Western blot detection with the same or different MYLK4 antibody can provide additional confirmation of specificity. This technique has been validated with HEK-293 cell lysates .

• Cross-Reactivity Testing: Although most MYLK4 antibodies are reported to be human-specific, systematic testing across species and related proteins within the MYLK family can help establish the boundaries of specificity .

• Peptide Competition Assays: Pre-incubating the antibody with the immunizing peptide or recombinant protein should abolish specific signal in positive samples, providing evidence of binding specificity.

• Multiple Antibody Concordance: Using multiple antibodies targeting different epitopes of MYLK4 and comparing their detection patterns can provide additional confidence in specificity.

How can MYLK4 antibodies be used to study protein-protein interactions and signaling pathways?

MYLK4 antibodies can be instrumental in unraveling protein-protein interactions and signaling networks through several advanced techniques:

Immunoprecipitation (IP) is particularly valuable for studying MYLK4's interaction partners. Using the established protocol (0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate), researchers can pull down MYLK4 along with its binding partners . Subsequent mass spectrometry analysis of co-immunoprecipitated proteins can reveal novel interaction partners and potential signaling complexes. This approach is especially relevant given MYLK4's membership in the CAMK Ser/Thr protein kinase family and its activation by Ca²⁺-calmodulin complexes .

For studying MYLK4 in signaling cascades, researchers can combine antibody-based detection with phosphorylation-specific antibodies to track activation states. Since MLCKs are activated by Ca²⁺-calmodulin complexes and require Mg²⁺ or Ca²⁺ as co-factors, antibody-based techniques can help elucidate how calcium signaling modulates MYLK4 activity in different cellular contexts .

Co-localization studies using immunofluorescence (IF) with MYLK4 antibodies alongside markers for subcellular compartments or potential interaction partners can provide spatial information about MYLK4's function. The antibodies have been validated for IF applications, making this approach feasible with appropriate controls .

For more dynamic studies, combining MYLK4 antibodies with proximity ligation assays (PLA) would allow researchers to visualize and quantify protein interactions in situ with high sensitivity, potentially revealing transient interactions that might be missed by conventional co-immunoprecipitation.

What considerations are important when using MYLK4 antibodies for tissue-specific expression studies?

When investigating tissue-specific expression of MYLK4, several important considerations emerge from the available data:

First, researchers should be aware of the validated tissue types where MYLK4 has been successfully detected. Positive IHC results have been reported in human heart tissue and human tonsillitis tissue , suggesting these as reliable positive controls. When examining new tissue types, including these validated samples as positive controls is advisable.

Antigen retrieval methods significantly impact the success of MYLK4 detection in tissue sections. The recommended protocol suggests using TE buffer at pH 9.0, with citrate buffer at pH 6.0 as an alternative . Comparative testing of both retrieval methods may be necessary for optimal results in different tissue types.

Dilution optimization is crucial, with recommended IHC dilutions ranging from 1:20 to 1:400 . A titration series should be performed for each new tissue type to determine optimal signal-to-noise ratio. Different tissues may require different antibody concentrations due to varying levels of target expression and potential cross-reactivity with tissue-specific proteins.

For quantitative analysis of expression levels across tissues, standardized protocols for staining, imaging, and quantification are essential. Researchers should consider automated staining platforms and image analysis software to ensure consistency when comparing MYLK4 expression between different tissues or experimental conditions.

When studying the two reported isoforms of MYLK4 produced by alternative splicing , researchers should verify which isoform(s) their antibody detects, as this may vary between antibody clones and could affect interpretation of tissue-specific expression patterns.

How do phosphorylation states affect MYLK4 antibody recognition?

The relationship between MYLK4 phosphorylation states and antibody recognition is an important consideration for researchers studying this kinase's regulation and activity:

Most commercial MYLK4 antibodies are generated against specific epitopes that may or may not include phosphorylation sites. For example, antibodies are produced using immunogens such as recombinant proteins corresponding to specific amino acid regions (e.g., Ala96~Ser353 or AA 286-314) . Researchers must determine whether their chosen antibody's epitope overlaps with known or potential phosphorylation sites to assess whether phosphorylation could affect recognition.

Since MYLK4 belongs to the CAMK Ser/Thr protein kinase family and is activated by Ca²⁺-calmodulin complexes , it likely undergoes phosphorylation events that could change its conformation and potentially mask or expose antibody epitopes. When studying MYLK4 activation, researchers should consider using phosphorylation-specific antibodies alongside total MYLK4 antibodies to distinguish between different activation states.

For applications requiring detection of specific phosphorylated forms, phospho-specific antibodies (if available) would be preferable. Alternatively, researchers could employ lambda phosphatase treatment of parallel samples to determine whether standard MYLK4 antibody recognition is affected by phosphorylation status.

When performing co-immunoprecipitation experiments to study MYLK4 interactions, researchers should consider how phosphorylation might affect not only antibody recognition but also protein-protein interactions. Preserving phosphorylation states through the use of phosphatase inhibitors in lysis buffers is advisable unless specifically studying dephosphorylated forms.

What are the most common causes of non-specific binding with MYLK4 antibodies and how can they be addressed?

Non-specific binding is a common challenge when working with antibodies, including those targeting MYLK4. Several causes and solutions should be considered:

Inadequate Blocking: Insufficient blocking can lead to high background. Researchers should optimize blocking conditions using 5% non-fat dry milk or 3-5% BSA in TBS-T for Western blotting applications. For IHC applications, normal serum from the species of the secondary antibody (typically goat or horse) at 2-10% concentration may be more effective .

Suboptimal Antibody Dilution: Too high antibody concentration often results in non-specific binding. Following the manufacturer's recommended dilution ranges (1:1000-1:4000 for WB, 1:100-1:400 for IHC) is a good starting point, but further optimization for each specific application and sample type is crucial .

Cross-Reactivity: MYLK4 antibodies may cross-react with related proteins in the myosin light chain kinase family. To address this, researchers can:

• Use knockout or knockdown controls to confirm specificity

• Perform peptide competition assays to verify that signal is specifically due to MYLK4 recognition

• Consider using monoclonal antibodies (if available) which typically offer higher specificity than polyclonal antibodies

Sample Preparation Issues: Incomplete denaturation for Western blotting or overfixation for IHC can lead to non-specific binding or poor results. For Western blotting, ensure complete denaturation of samples, and for IHC, optimize fixation times and conditions. For IHC applications with MYLK4 antibodies, specific antigen retrieval methods using TE buffer pH 9.0 or alternatively citrate buffer pH 6.0 are recommended .

Secondary Antibody Cross-Reactivity: This can be addressed by using more highly cross-adsorbed secondary antibodies and including controls where primary antibody is omitted but secondary antibody is included.

Washing Conditions: Insufficient washing can leave residual unbound antibody. For MYLK4 antibody applications, increasing the number and duration of washing steps with TBS-T or PBS-T can significantly reduce background.

How should researchers address discrepancies in MYLK4 detection between different experimental techniques?

When researchers encounter discrepancies in MYLK4 detection across different techniques (e.g., positive WB but negative IHC), several methodological approaches can help resolve these inconsistencies:

Antibody Epitope Accessibility: Different techniques expose different epitopes. Western blotting completely denatures proteins, exposing most epitopes, while IHC maintains some native structure and potentially masks certain epitopes. Researchers should verify which techniques an antibody has been validated for. For example, some MYLK4 antibodies are validated for WB, IP, IHC, and ELISA , while others may only be validated for a subset of these applications .

Sample Preparation Differences: For inconsistencies between techniques, researchers should examine how sample preparation affects MYLK4 detection:

• For Western blotting: optimize lysis buffers, denaturation conditions, and loading amounts

• For IHC: test different fixatives, fixation times, and antigen retrieval methods (TE buffer pH 9.0 is recommended for MYLK4, with citrate buffer pH 6.0 as an alternative)

• For IP: evaluate lysis conditions that preserve protein-protein interactions while efficiently extracting MYLK4

Antibody Selection: When facing discrepancies, using multiple antibodies targeting different MYLK4 epitopes can help determine whether the issue is technique-dependent or epitope-dependent. The search results indicate various MYLK4 antibodies targeting different regions, such as AA 96-353, AA 3-100, and AA 286-314 (C-term) .

Control Inclusion: For any technique showing negative results, include positive controls where MYLK4 has been reliably detected. For WB, HEK-293 and L02 cells have shown positive detection; for IHC, human heart tissue and tonsillitis tissue are recommended positive controls .

Expression Level Sensitivity: Different techniques have different detection thresholds. Western blotting with chemiluminescence can be more sensitive than standard IHC. When expression levels are low, consider signal amplification methods for IHC or using more sensitive detection systems for Western blotting.

Isoform Specificity: Since MYLK4 has two reported isoforms from alternative splicing , discrepancies might arise from isoform-specific detection. Researchers should determine which isoform(s) their antibody recognizes and whether different isoforms predominate in different experimental systems.

What are the recommended protocols for detecting low abundance MYLK4 in tissues or cells?

Detecting low abundance proteins like MYLK4 in certain tissues or cell types requires specialized approaches to enhance sensitivity while maintaining specificity:

For Western Blotting:

• Increase protein loading (50-100 μg per lane)

• Use high-sensitivity chemiluminescent substrates or fluorescent detection systems

• Optimize primary antibody incubation by extending to overnight at 4°C with dilutions at the more concentrated end of the recommended range (e.g., 1:500-1:1000)

• Consider membrane transfer optimization: use PVDF membranes (higher protein binding capacity than nitrocellulose) and transfer at lower voltage for longer periods

• Employ protein concentration techniques such as immunoprecipitation prior to Western blotting

• Use signal enhancement systems such as biotin-streptavidin amplification

For Immunohistochemistry:

• Optimize antigen retrieval thoroughly, testing both recommended methods (TE buffer pH 9.0 and citrate buffer pH 6.0)

• Use signal amplification systems such as tyramide signal amplification (TSA)

• Consider more sensitive detection systems like polymer-based detection rather than traditional ABC methods

• Increase primary antibody concentration (1:20-1:100 range) and extend incubation times to overnight at 4°C

• Reduce background with extended blocking (3-5% BSA or appropriate serum for 1-2 hours) and washing steps

• For particularly challenging samples, consider using frozen sections rather than paraffin-embedded tissues to better preserve epitopes

For Immunoprecipitation:

• Increase starting material (3-5 mg of total protein)

• Extend antibody binding time to overnight at 4°C

• Use the higher end of the recommended antibody amount (3-4 μg) for maximum pull-down efficiency

• Pre-clear lysates thoroughly to reduce non-specific binding

• Optimize lysis buffers to ensure efficient MYLK4 extraction while preserving the epitope structure

How can researchers optimize MYLK4 antibodies for multiplexed imaging studies?

Multiplexed imaging to simultaneously detect MYLK4 alongside other proteins requires careful planning and optimization:

Antibody Selection for Multiplexing:

• Choose MYLK4 antibodies from different host species than other target antibodies to avoid cross-reactivity (rabbit polyclonal MYLK4 antibodies are most common, so pair with mouse, goat, or rat antibodies for other targets)

• If using multiple rabbit antibodies, consider direct conjugation to distinct fluorophores or sequential detection with tyramide signal amplification

• Verify that the MYLK4 antibody has been validated for immunofluorescence applications

Optimization Steps:

• Test each antibody individually before combining to establish optimal working dilutions and conditions

• For MYLK4, start with dilutions recommended for IHC (1:100-1:400) and adjust based on signal intensity

• Carefully select fluorophores with minimal spectral overlap

• Include appropriate controls:

  • Single-stained samples for each antibody to confirm specificity

  • Secondary-only controls to check for non-specific binding

  • Autofluorescence controls, especially important in tissues like heart where MYLK4 has been detected

Advanced Multiplexing Approaches:

• For highly complex multiplexing, consider cyclic immunofluorescence or mass cytometry methods

• Sequential staining protocols can be employed when antibodies from the same species must be used

• For co-localization studies with MYLK4, prioritize targets that might interact with MYLK4 based on its function in the CAMK Ser/Thr protein kinase family

• When studying MYLK4 activation, include antibodies against calcium signaling components, given that MLCKs are activated by Ca²⁺-calmodulin complexes

Image Acquisition and Analysis Considerations:

• Use spectral unmixing algorithms to separate overlapping fluorophore signals

• Employ consistent exposure settings when comparing MYLK4 expression across different conditions

• Consider advanced analysis methods such as proximity analysis to study potential interactions between MYLK4 and other proteins

• For quantitative comparisons, include fluorescent standards to normalize signal intensities across experiments

How can MYLK4 antibodies be used to investigate its role in human disease models?

MYLK4 antibodies offer valuable tools for exploring this protein's potential roles in various disease states, particularly in cardiac and muscle-related conditions given its function as a myosin light chain kinase:

Cardiac Disease Models:
Since MYLK4 shows positive detection in human heart tissue , researchers can utilize these antibodies to investigate MYLK4's expression and localization in various cardiac pathologies. Using IHC with the recommended dilutions (1:100-1:400) and antigen retrieval methods (TE buffer pH 9.0), researchers can compare MYLK4 expression patterns between normal and diseased cardiac tissues. Western blotting of cardiac tissue lysates using dilutions of 1:1000-1:4000 can provide quantitative comparisons of expression levels.

Functional Studies in Disease Models:
For functional investigations, MYLK4 antibodies can be used in combination with knockdown/knockout approaches. Published applications indicate that these antibodies are suitable for KD/KO experimental systems , allowing researchers to validate targeting efficiency and explore phenotypic consequences in disease-relevant cell or animal models.

Signaling Pathway Analysis:
Since MYLK4 belongs to the CAMK Ser/Thr protein kinase family and is activated by Ca²⁺-calmodulin complexes , researchers can employ these antibodies to investigate calcium signaling dysregulation in disease states. Immunoprecipitation approaches using 0.5-4.0 μg of antibody per 1.0-3.0 mg lysate can help identify disease-specific interaction partners or post-translational modifications.

Translational Research Applications:
For potential biomarker studies, researchers can optimize MYLK4 antibodies for applications such as tissue microarray analysis, allowing high-throughput evaluation of MYLK4 expression across large patient cohorts. Similarly, these antibodies could be adapted for flow cytometry to analyze MYLK4 in circulating cells or cell suspensions derived from disease-relevant tissues.

What are the considerations for using MYLK4 antibodies in phosphoproteomic analyses?

Incorporating MYLK4 antibodies into phosphoproteomic studies requires specific technical considerations to effectively capture and analyze phosphorylation dynamics:

Phosphorylation-State Specificity:
Standard MYLK4 antibodies (as described in the search results) recognize total protein rather than specific phosphorylated forms . For phosphoproteomic analyses, researchers should determine whether their antibody's epitope includes or is affected by phosphorylation sites. When specific phospho-MYLK4 antibodies are unavailable, researchers can employ strategies such as:

• Immunoprecipitating total MYLK4 followed by phospho-specific detection using generic phospho-Ser/Thr antibodies

• Using lambda phosphatase-treated versus untreated samples to differentiate phosphorylated from non-phosphorylated forms

Sample Preparation for Phosphoproteomics:
When studying MYLK4 phosphorylation, sample preparation should include:

• Rapid sample collection and processing to preserve phosphorylation states

• Inclusion of phosphatase inhibitors in all buffers

• Consideration of phosphopeptide enrichment methods (TiO₂, IMAC, etc.) when analyzing MYLK4 phosphorylation by mass spectrometry

Integration with Mass Spectrometry:
For comprehensive phosphoproteomic analysis, researchers can:

• Use MYLK4 antibodies (0.5-4.0 μg per IP reaction) to immunoprecipitate the protein from relevant samples

• Subject immunoprecipitated material to tryptic digestion and phosphopeptide enrichment

• Analyze resulting peptides by LC-MS/MS to identify specific phosphorylation sites

• Compare phosphorylation profiles under different conditions (e.g., calcium stimulation, disease states)

Validation Approaches:
Any phosphorylation sites identified through discovery proteomics should be validated using complementary methods such as:

• Targeted mass spectrometry (MRM/PRM)

• Site-specific phospho-antibodies (if available)

• Functional assays examining the consequences of phospho-mimetic or phospho-null mutations at identified sites

Kinase Activity Considerations:
Since MYLK4 itself is a kinase activated by Ca²⁺-calmodulin complexes , researchers should consider both MYLK4 as a target of phosphorylation and as a mediator of phosphorylation events. Experimental designs incorporating both perspectives will provide more comprehensive understanding of MYLK4's role in signaling networks.

What best practices should researchers follow to ensure reproducible results with MYLK4 antibodies?

To ensure reproducible and reliable results when working with MYLK4 antibodies, researchers should adhere to the following best practices:

Antibody Validation and Documentation:

• Verify antibody specificity through multiple validation methods, including positive and negative controls (MYLK4 antibodies have shown positive detection in HEK-293 cells, L02 cells, human heart tissue, and human tonsillitis tissue)

• Document complete antibody information including catalog number, lot number, host species, clonality, and epitope information in all publications and lab records

• Consider using multiple antibodies targeting different MYLK4 epitopes to confirm results, especially for novel findings

Standardized Protocols:

• Establish detailed, written protocols for each application with specific attention to:

  • Sample preparation methods

  • Antibody dilutions (follow recommended ranges: WB 1:500-1:5000, IHC 1:20-1:400, IP 0.5-4.0 μg)

  • Incubation times and temperatures

  • Buffer compositions

  • Antigen retrieval methods for IHC (TE buffer pH 9.0 or citrate buffer pH 6.0)

• Include all relevant controls in every experiment

• Establish quantification methods that are objective and reproducible

Experimental Design Considerations:

• Determine appropriate sample sizes through power analysis

• Incorporate biological replicates across different cell preparations or specimens

• Include technical replicates to assess method reproducibility

• Design experiments to minimize batch effects

• Consider blinding analysis where appropriate, especially for IHC scoring

Data Reporting Standards:

• Report all experimental conditions in detail

• Include representative images of all data

• Present quantitative data with appropriate statistical analysis

• Provide access to original, unmodified blot images

• Disclose any image processing or adjustments applied

Long-term Considerations:

• Monitor antibody performance across lots

• Periodically revalidate antibodies, especially after receiving new lots

• Consider creating internal reference standards for long-term studies

• Store antibodies according to manufacturer recommendations (-20°C, avoiding repeated freeze-thaw cycles)

By following these best practices, researchers can maximize the reliability and reproducibility of their MYLK4 antibody-based experiments and facilitate comparison of results across different studies and laboratories.

What emerging technologies might enhance the utility of MYLK4 antibodies in future research?

Several emerging technologies have the potential to significantly expand the applications and improve the performance of MYLK4 antibodies in research settings:

Single-Cell Technologies:
Adapting MYLK4 antibodies for single-cell proteomics and imaging techniques would enable researchers to investigate cell-to-cell variability in MYLK4 expression and activation. Technologies such as mass cytometry (CyTOF), imaging mass cytometry, and single-cell Western blotting could be optimized using the established dilution ranges (1:500-1:5000 for protein detection) to analyze MYLK4 at single-cell resolution.

Spatially Resolved Proteomics:
Emerging spatial proteomics technologies could be combined with MYLK4 antibodies to map the protein's distribution within tissues with unprecedented resolution. Techniques such as Digital Spatial Profiling (DSP), Multiplexed Ion Beam Imaging (MIBI), and various in situ sequencing approaches could leverage validated MYLK4 antibodies to reveal spatial relationships between MYLK4 expression and tissue architecture, particularly in tissues where positive detection has been demonstrated (heart, tonsil) .

Proximity Labeling Approaches:
Antibody-directed proximity labeling techniques could enhance our understanding of MYLK4's interaction network. By conjugating promiscuous labeling enzymes (BioID, APEX) to MYLK4 antibodies or using antibodies to immunoprecipitate MYLK4 for subsequent proximity labeling studies, researchers could map the protein's immediate microenvironment in living cells.

Nanobody and Alternative Scaffold Development:
Developing nanobodies or alternative binding scaffolds against MYLK4 could overcome limitations of traditional antibodies, providing smaller probes with potentially better tissue penetration and access to sterically hindered epitopes. These could be particularly valuable for super-resolution microscopy applications studying MYLK4 localization at the nanoscale.

Antibody Engineering:
Genetic fusion of MYLK4 antibody fragments with various reporter proteins or functional domains could generate novel research tools. For example, creating split fluorescent protein complementation systems involving MYLK4-targeted antibody fragments could enable live-cell visualization of MYLK4 interactions or conformational changes related to its activation by Ca²⁺-calmodulin complexes .

Integrated Multi-Omics Approaches: Combining MYLK4 antibody-based proteomics with transcriptomics, metabolomics, and functional genomics will provide systems-level understanding of MYLK4 biology. Computational integration of data from MYLK4 antibody studies with other -omics datasets will help position this protein within broader biological networks and potentially reveal unexpected functions or regulatory mechanisms.