Pan Methylated Lysine Monoclonal Antibody

What is a Pan Methylated Lysine Monoclonal Antibody and how does it differ from methylsite-specific antibodies?

Pan Methylated Lysine Monoclonal Antibodies recognize methylated lysine residues regardless of the surrounding amino acid sequence, detecting this post-translational modification across different proteins. These antibodies bind to the methylated lysine moiety itself, making them valuable for global methylation studies.

The development of methylsite-specific monoclonal antibodies remains challenging compared to other modification-specific antibodies (like phosphosite-specific antibodies), largely because the chemical changes introduced by methylation (-CH3 groups) are relatively subtle .

What molecular mechanisms enable Pan Methylated Lysine Monoclonal Antibodies to recognize their targets?

Pan Methylated Lysine Monoclonal Antibodies typically recognize methylated lysine through specialized binding domains containing aromatic cages. X-ray crystallography and molecular dynamics simulations of methylated lysine-specific antibodies reveal that these aromatic cages create a hydrophobic pocket that accommodates the methylated lysine residue .

The stability of binding varies between antibodies, with high-affinity antibodies (like C9, E6, and D6 described in research studies) maintaining stable interactions with the methylated lysine within the aromatic cage throughout molecular dynamics simulations. Lower affinity antibodies (like F9 in the referenced study) show dynamic movement of the methylated lysine, sometimes completely exiting the aromatic cage .

The recognition mechanism often involves:

• Initial binding through interactions with the peptide backbone

• Stabilization of the methylated lysine within an aromatic cage formed by residues in the antibody's complementarity determining regions (CDRs)

• Additional interactions with surrounding amino acids that may influence specificity

These structural features explain why some antibodies display preferences for specific methylation states (mono-, di-, or tri-methylation) .

How do Pan Methylated Lysine Monoclonal Antibodies recognize different methylation states?

Pan Methylated Lysine Monoclonal Antibodies typically show varying affinities for different methylation states (mono-, di-, and tri-methylation) of lysine residues. Surface plasmon resonance (SPR) analysis demonstrates that the affinity of these antibodies generally increases with the degree of methylation, although this pattern varies among different antibody clones .

For example, in studies of methylated lysine-specific antibodies:

• Antibody clones like C9 and D6 showed higher specificity toward trimethylated lysine compared to mono- and di-methylated forms

• The preference for higher methylation states likely results from increased hydrophobicity and van der Waals interactions with the antibody's aromatic cage

This differential recognition explains why Western blot patterns may show varying intensities when probing for proteins with different methylation states. Researchers should be aware of this variable affinity when interpreting results, especially in comparative studies examining changes in methylation status .

What are the optimal conditions for using Pan Methylated Lysine Monoclonal Antibody in Western blot applications?

For optimal Western blot results with Pan Methylated Lysine Monoclonal Antibodies, follow these methodological guidelines:

Sample Preparation:

• Extract proteins using RIPA buffer or similar extraction methods that preserve post-translational modifications

• Include deacetylase inhibitors, protease inhibitors, and phosphatase inhibitors in extraction buffers

• For cell lysates, dilute appropriately in RIPA buffer before immunoprecipitation

Antibody Dilution and Incubation:

• Use dilutions between 1:1,000-3,000 for Western blot applications

• For mouse monoclonal antibodies (like those from ELK Biotechnology), dilutions of 1:1,000-2,000 are recommended

• Incubate the membrane with primary antibody overnight at 4°C for optimal signal-to-noise ratio

Detection System:

• Use HRP-conjugated secondary antibodies at 1:5,000 dilution

• Develop using ECL technique with appropriate exposure time (approximately 20 minutes has been effective in published protocols)

Expected Results:

• Bands corresponding to methylated histones appear at 14-17 kDa

• Other methylated proteins will appear at their respective molecular weights

• Be aware that some non-specific bands may appear, requiring careful validation

For optimal specificity, include proper controls (unmethylated proteins, methylation-deficient mutants) to validate the specificity of detected bands.

How can Pan Methylated Lysine Monoclonal Antibody be used for immunoprecipitation of methylated proteins?

Immunoprecipitation (IP) with Pan Methylated Lysine Monoclonal Antibodies enables enrichment of methylated proteins from complex samples. The following methodological approach has been validated in published research:

IP Protocol:

• Incubate the antibody (approximately 1-5 μg) with Protein G beads under agitation for 10 minutes

• Add cell extract lysate diluted in RIPA buffer

• Incubate for an additional 10 minutes under agitation

• Wash beads thoroughly to remove non-specific interactions

• Elute proteins by adding SDS loading buffer and incubating at 70°C for 10 minutes

• Analyze the eluted proteins by SDS-PAGE and Western blotting

Validation Strategy:
To confirm specificity of the immunoprecipitated proteins, perform competitive assays using methylated peptides to quench antibody activity. Published studies demonstrate that immunizing peptides like Human Histone H3 (di methyl K4) peptide, Human Histone H3 (di methyl K9) peptide, and Human Histone H3 (di methyl K27) peptide can effectively block antibody recognition of the corresponding methylation sites .

This approach allows researchers to verify which specific methylated proteins are being enriched and can help distinguish true targets from non-specific binding events.

What methodological approaches should be used for immunohistochemistry with Pan Methylated Lysine Monoclonal Antibody?

For successful immunohistochemistry (IHC) using Pan Methylated Lysine Monoclonal Antibodies, the following methodological parameters should be optimized:

Tissue Preparation:

• For formalin-fixed paraffin-embedded (FFPE) tissues, effective antigen retrieval is critical

• Heat-mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 20 minutes has been validated for pan methylated lysine detection

Antibody Concentration and Incubation:

• For mouse monoclonal antibodies, use dilutions of 1:200-500 for IHC applications

• When using specific antibodies like ab7315, a concentration of 1 μg/ml with 15-minute incubation at room temperature has shown good results

Detection System:

• HRP-conjugated compact polymer systems with DAB as the chromogen provide effective visualization

• Counterstain with hematoxylin and mount with appropriate medium (such as DPX)

Controls and Validation:

• Include positive controls (tissues with known methylation patterns)

• Negative controls should omit primary antibody

• For validation, perform peptide competition assays using methylated peptides

The detection of methylated proteins in IHC typically reveals nuclear staining patterns, reflecting the abundance of methylated histones, though cytoplasmic staining may also be observed for non-histone methylated proteins. Careful evaluation of staining patterns alongside appropriate controls is essential for accurate interpretation.

How can immunofluorescence experiments be optimized when using Pan Methylated Lysine Monoclonal Antibody?

For immunocytochemistry/immunofluorescence (ICC/IF) applications with Pan Methylated Lysine Monoclonal Antibodies, the following methodological approach offers optimal results:

Cell Fixation and Permeabilization:

• Fix cells with 100% methanol for 5 minutes

• Permeabilize and block non-specific binding with 1% BSA/10% normal serum/0.3M glycine in 0.1% PBS-Tween for 1 hour

Antibody Incubation:

• Incubate cells with the primary antibody (5 μg/ml) overnight at 4°C

• Wash thoroughly with PBS-Tween

• Apply fluorophore-conjugated secondary antibody (such as DyLight® 488 anti-rabbit IgG) at 1:1,000 dilution for 1 hour

Co-staining and Nuclear Visualization:

• Membrane visualization: Alexa Fluor® 594 WGA at 1:200 dilution (1 hour)

• Nuclear counterstaining: DAPI at 1.43 μM concentration

Image Acquisition:

• Use confocal microscopy for optimal resolution

• Capture z-stacks to assess nuclear vs. cytoplasmic distribution

• Employ appropriate filter settings to minimize bleed-through

Methylated lysine staining typically shows strong nuclear localization due to histone methylation, although cytoplasmic staining may also be observed depending on the cell type and physiological state. Compare staining patterns between different cell types or treatment conditions to identify biologically relevant changes in methylation.

How do monoclonal antibody cocktails compare to single polyclonal antibodies for methylated lysine detection?

Monoclonal antibody cocktails and polyclonal antibodies for methylated lysine detection show distinct advantages and complementary detection profiles. Here's a methodological comparison based on research findings:

Detection Profiles:
A comparative study between monoclonal antibody cocktails and polyclonal antibodies for acetylated lysine (which provides relevant insights for methylated lysine detection) demonstrated that:

• The overlap of identified modified residues was limited to only 28.8-37.6% between the two approaches

• Each method identified a unique subset of modified sites

• Replicate runs using the same approach showed higher consistency (52.5-58.9% overlap)

Advantages of Monoclonal Antibody Cocktails:

• Composed of antibodies raised against diverse antigens containing the modified lysine

• Each monoclonal in the cocktail targets the modification in different sequence contexts

• More easily standardized and reproducible between batches

• Potentially unlimited supply compared to polyclonal antibodies

Statistical Verification:
The difference in detection profiles between monoclonal cocktails and polyclonal antibodies is statistically significant (p-values ranging from 0.038 to <0.001), even when adjusting for run-to-run variation .

This suggests that for comprehensive methylation profiling, researchers should consider using both approaches as complementary methods rather than alternatives. The ideal strategy may be to perform parallel experiments with both antibody types to maximize coverage of the methylated proteome.

What experimental approaches can verify the specificity of Pan Methylated Lysine Monoclonal Antibody results?

To verify the specificity of Pan Methylated Lysine Monoclonal Antibody results, implement these methodological validation strategies:

Peptide Competition Assays:

• Pre-incubate the antibody with specific methylated peptides (e.g., Human Histone H3 di-methyl peptides)

• If the antibody is specific, pre-incubation will quench the signal for corresponding methylated proteins

• Compare results with and without peptide competition to identify specific bands

Mutagenesis Validation:

• Generate methylation site mutants (e.g., K→A or K→R) of the protein of interest

• Express wild-type and mutant proteins in appropriate cell systems

• Compare antibody detection between wild-type and mutant proteins

• A significant reduction in signal with the mutant confirms specificity

Methyltransferase Dependency:

• Manipulate the expression of the methyltransferase responsible for the modification

• For example, studies with SMYD3 methyltransferase showed MAP3K2 methylation at K260 was dependent on SMYD3 expression

• Detection of methylated proteins should correlate with methyltransferase levels

Molecular Weight Confirmation:

• For histone methylation, verify that detected bands correspond to expected molecular weights (H3 = 17 kDa, H4 = 14 kDa)

• Non-histone methylated proteins should align with their predicted molecular weights

Implementing these validation approaches helps distinguish genuine methylation signals from potential cross-reactivity, ensuring reliable interpretation of experimental results.

How does the antibody's recognition vary between mono-, di-, and tri-methylated lysine residues?

Pan Methylated Lysine Monoclonal Antibodies typically display differential recognition of mono-, di-, and tri-methylated lysine residues, which is important to understand when interpreting experimental results:

Affinity Gradient:
Surface plasmon resonance (SPR) analysis demonstrates that antibody affinity generally increases with higher methylation states, with a preference pattern of:
Trimethylation > Dimethylation > Monomethylation

Antibody-Specific Variation:
This methylation state dependency is not identical among different antibody clones:

• Some antibodies (like C9 and D6 in published studies) show stronger preference for trimethylated lysine

• Other antibodies exhibit more similar affinities across different methylation states

• These differences affect the interpretation of Western blots and other detection methods

Structural Basis for Preference:
The preferential recognition of higher methylation states is attributed to:

• Enhanced hydrophobic interactions with the aromatic cage of the antibody

• More stable binding of trimethylated lysine within the recognition pocket

• Molecular dynamics simulations showing trimethylated lysine maintains position within the aromatic cage more consistently than lesser methylated forms

This differential recognition has practical implications for experimental design. When studying proteins with mixed methylation states, researchers should be aware that signal intensity may reflect both abundance and methylation state preference of the antibody, potentially requiring complementary analytical approaches for complete characterization.

What are the common causes of false positive or false negative results when using Pan Methylated Lysine Monoclonal Antibody?

When using Pan Methylated Lysine Monoclonal Antibodies, several factors can lead to false positive or negative results. Understanding these issues helps researchers implement appropriate controls and optimization strategies:

Causes of False Positives:

• Cross-reactivity with similar modifications:

  • Some antibodies may detect acetylated lysine or other PTMs with similar chemical properties

  • Validation strategy: Perform peptide competition assays with specifically modified peptides

• Non-specific binding to hydrophobic protein regions:

  • The aromatic cage that recognizes methylated lysine may bind non-specifically to hydrophobic patches

  • Validation strategy: Include methylation-deficient mutants (K→A) as negative controls

• Off-target recognition of similar sequence contexts:

  • Western blots may show bands corresponding to proteins with similar sequences surrounding methylated lysine

  • Example: Studies observed SMYD3-dependent bands beyond the expected MAP3K2 target

  • Validation strategy: Use multiple antibody clones to confirm results

Causes of False Negatives:

• Insufficient antigen retrieval in fixed samples:

  • For FFPE tissues, inadequate antigen retrieval can mask methylation sites

  • Optimization strategy: Test different antigen retrieval methods (heat-mediated with citrate buffer has shown success)

• Antibody preference for specific methylation states:

  • An antibody with strong preference for trimethylation might not detect mono-methylated proteins

  • Optimization strategy: Use antibody cocktails with complementary specificities

• Epitope masking by protein-protein interactions:

  • Methylated lysines involved in protein interactions may be inaccessible to antibodies

  • Optimization strategy: Use denaturing conditions for Western blots and optimize extraction protocols

Implementing proper controls, including peptide competitions and methylation-deficient mutants, is essential for distinguishing true signals from artifacts and ensuring reliable experimental outcomes.

How can researchers optimize immunoprecipitation protocols to improve methylated protein enrichment?

Optimizing immunoprecipitation (IP) protocols for methylated protein enrichment requires attention to several methodological details:

Buffer Optimization:

• Include deacetylase inhibitors, protease inhibitors, and phosphatase inhibitors in lysis buffers

• RIPA buffer has been validated for effective extraction while preserving methylation modifications

• Adjust salt concentration (150-300 mM NaCl) to balance specificity and yield

Antibody Immobilization Strategy:

• Pre-incubation method: Incubate antibody with Protein G beads for 10 minutes before adding lysate

• Direct capture method: Add antibody directly to lysate, then capture with Protein G beads

• Comparative studies suggest the pre-incubation method yields cleaner results with less non-specific binding

Incubation Parameters:

• Optimal lysate incubation time: 10 minutes under agitation at room temperature

• Extended incubation (2-4 hours) may increase yield but can introduce non-specific binding

• Temperature control (4°C) helps preserve protein integrity during longer incubations

Sequential IP Approach:
For comprehensive coverage, consider a sequential IP strategy:

• Perform first IP with monoclonal antibody cocktail

• Collect unbound fraction

• Perform second IP on unbound fraction using polyclonal antibody

• This approach captures complementary subsets of methylated proteins

Elution Optimization:

• Standard elution: SDS loading buffer at 70°C for 10 minutes

• Native elution (for functional studies): Competition with excess methylated peptide

• Acidic glycine elution (pH 2.5-3.0) can preserve antibody for reuse

These optimizations significantly improve the specificity and yield of methylated protein enrichment, enabling more comprehensive analysis of the methyl-proteome.

What sample preparation considerations are critical when working with Pan Methylated Lysine Monoclonal Antibody?

Proper sample preparation is crucial for successful detection of methylated proteins using Pan Methylated Lysine Monoclonal Antibodies. The following methodological considerations help preserve methylation status and optimize antibody performance:

Cell and Tissue Lysis:

Protein Denaturation Considerations:

• For Western blot applications, complete denaturation with SDS and heat (70°C for 10 minutes) improves epitope accessibility

• Reducing conditions are recommended for optimal results

Antigen Retrieval for Fixed Tissues:

• For FFPE sections, heat-mediated antigen retrieval with sodium citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 minutes is effective

• Microwave or pressure cooker methods may yield superior results compared to water bath methods

Storage Considerations:

• Antibodies should be stored at -20°C for maximum stability

• Avoid repeated freeze-thaw cycles that can reduce activity

• For long-term storage, aliquoting is recommended

Buffer Compatibility:

• Standard PBS with 0.02% sodium azide and 50% glycerol (pH 7.4) maintains antibody stability

• For IP applications, verify compatibility between antibody buffer and IP buffer systems

Attention to these sample preparation details significantly improves reproducibility and sensitivity when working with Pan Methylated Lysine Monoclonal Antibodies across different experimental applications.

How can Pan Methylated Lysine Monoclonal Antibody be used in combination with mass spectrometry for comprehensive methyl-proteome analysis?

Combining Pan Methylated Lysine Monoclonal Antibodies with mass spectrometry creates a powerful approach for comprehensive methyl-proteome analysis. The following methodological workflow maximizes coverage and confidence in methylation site identification:

Enrichment Strategy:

• Use a monoclonal antibody cocktail approach to capture diverse methylated peptides

• Perform parallel enrichments with different antibody types (monoclonal cocktail and polyclonal)

• This complementary approach significantly increases the coverage of the methyl-proteome, as studies with acetylated lysine antibodies showed limited overlap (28.8-37.6%) between antibody types

MS/MS Analysis Optimization:

• High-resolution Fourier transform mass spectrometry significantly outperforms ion trap CID methods

• HCD (Higher-energy Collisional Dissociation) fragmentation with Orbitrap detection yields considerably more methylation sites than CID with ion trap detection

• This technical difference is crucial for comprehensive methylation site identification

Statistical Validation:
To ensure confidence in identified methylation sites:

• Perform multiple biological replicates (minimum of three recommended)

• Calculate overlap between replicate runs (typical values: 52.5-58.9% between replicates)

• Apply appropriate statistical filters to distinguish true methylation sites from false positives

Comparative Analysis Strategy:
For studies examining changes in methylation status:

• Use SILAC or TMT labeling to enable quantitative comparison

• Account for antibody affinity bias toward higher methylation states

• Normalize data appropriately to distinguish changes in methylation from changes in protein abundance

This integrated approach leverages the specificity of antibody-based enrichment with the analytical power of mass spectrometry, enabling comprehensive characterization of the methyl-proteome even for low-abundance modifications.

What approaches can be used to study the structural basis of antibody recognition of methylated lysine residues?

Understanding the structural basis of methylated lysine recognition by antibodies provides insights for both basic science and antibody engineering. The following methodological approaches have proven effective:

X-ray Crystallography:

• Generate Fab fragments from pan methylated lysine monoclonal antibodies

• Co-crystallize Fab fragments with methylated peptide antigens

• Solve crystal structures to reveal atomic-level details of the antibody-antigen interface

• This approach has successfully revealed the aromatic cages that form the methylated lysine binding pocket

Molecular Dynamics Simulations:

• Conduct multiple 400-ns molecular dynamics simulations for each Fab-peptide complex

• Calculate RMSD values of the NZ atoms of methylated lysine after superposing aromatic residues

• Monitor stability of the methylated lysine within the aromatic cage

• Calculate solvent accessible surface area of the methylated lysine residue

These simulations reveal dynamics that static crystal structures cannot, showing that:

• High-affinity antibodies (like C9, E6, D6) maintain stable binding of methylated lysine

• Lower-affinity antibodies (like F9) show dynamic movement and occasional ejection of methylated lysine from the binding pocket

Structure-Function Analysis:
Correlate structural observations with functional assays:

• SPR analysis to determine binding kinetics and affinities

• Compare antibodies with different aromatic cage configurations

• Evaluate specificity toward different methylation states (mono-, di-, tri-methylation)

This comprehensive structural analysis has practical applications for:

• Designing improved methylation-specific antibodies

• Engineering antibodies with tailored specificity for particular methylation states

• Developing rational strategies for generating methylsite-specific antibodies

The combined approach of crystallography and molecular dynamics provides predictive power for antibody specificity, potentially enabling in silico screening of antibody variants .

How can researchers investigate the relationship between histone and non-histone protein methylation using Pan Methylated Lysine Monoclonal Antibody?

Investigating the relationship between histone and non-histone protein methylation requires strategic experimental design using Pan Methylated Lysine Monoclonal Antibodies. The following methodological approach facilitates such comparative studies:

Subcellular Fractionation Strategy:

• Separate nuclear, cytoplasmic, and chromatin-bound fractions

• Perform Western blot analysis with pan methylated lysine antibodies on each fraction

• This approach distinguishes methylation patterns in different cellular compartments

• Histone methylation appears predominantly in nuclear/chromatin fractions (14-17 kDa bands)

• Non-histone methylation can be observed across different fractions

Methyltransferase Manipulation:

• Modulate expression of specific methyltransferases (e.g., SMYD3)

• Monitor changes in both histone and non-histone methylation patterns

• Correlate methyltransferase activity with specific targets

For example, studies with SMYD3 demonstrated:

• SMYD3-dependent methylation of the non-histone protein MAP3K2 at K260

• Simultaneous effects on selected histone methylation sites

• This approach reveals shared enzymatic regulation between histone and non-histone targets

Temporal Analysis During Cellular Transitions:

• Track methylation changes during processes like differentiation or stress response

• Compare kinetics of histone vs. non-histone methylation changes

• This approach reveals whether histone and non-histone methylation are coordinated or independent events

Co-immunoprecipitation Studies:

• Immunoprecipitate methylated histones

• Probe for associated non-histone proteins

• Reverse IP: Immunoprecipitate specific non-histone proteins and probe for methylation

• This approach identifies potential physical interactions between methylated histones and non-histone proteins

These methodological strategies provide insights into whether histone and non-histone methylation represent parallel, independent modification systems or coordinated regulatory networks with shared enzymatic machinery and biological functions.

What strategies can be employed to develop improved methylation site-specific monoclonal antibodies?

Developing improved methylation site-specific monoclonal antibodies remains challenging but critical for advancing protein methylation research. The following methodological approaches represent cutting-edge strategies:

Immunization Strategy Optimization:

• Conjugate methylated peptides to carrier proteins that enhance immunogenicity

• Use rabbits rather than mice for immunization, as rabbit monoclonal antibodies generally possess higher affinity (picomolar range) compared to mouse antibodies

• Implement strategic immunization schedules with appropriate adjuvants to maximize immune response

Selection Technology Integration:

• Combine traditional immunization with phage display technology

• Construct antibody libraries from immunized animals

• Implement iterative rounds of panning with stringent selection pressure

• This selection-based approach has advantages over screening-based methods (like hybridoma) for obtaining highly specific clones

Structural Biology-Guided Design:

• Analyze crystal structures of successful methylation-specific antibodies

• Identify critical features of the aromatic cage that recognizes methylated lysine

• Engineer complementarity determining regions (CDRs) to optimize the aromatic cage configuration

• Molecular dynamics simulations can predict antibody specificity toward methylation, guiding rational design

Cocktail Development Strategy:

• Generate multiple monoclonal antibodies against the same methylation site in different sequence contexts

• Characterize individual clones for specificity and affinity

• Create optimized cocktails of complementary antibodies

• This approach significantly increases coverage compared to single antibodies

The integration of these approaches represents a significant advancement over traditional methods, potentially yielding antibodies with:

• Superior specificity for particular methylation sites

• Enhanced discrimination between methylation states

• Improved performance across diverse applications

• Greater batch-to-batch consistency

These methodological innovations address the historical challenges in creating reliable methylation-specific antibodies, potentially facilitating the same type of research expansion that phospho-specific antibodies enabled for phosphorylation studies .