Phospho-MAK (Y159) Antibody

What is MAK kinase and why is Y159 phosphorylation important?

MAK (Male germ cell-Associated Kinase) is a serine/threonine protein kinase belonging to the MAP kinase family. It contains a conserved TDY motif (Threonine-Aspartate-Tyrosine) at positions 157-159. Phosphorylation at Y159 occurs as part of a dual phosphorylation event on this TDY motif that is crucial for full MAK kinase activity. Studies have shown that when the TDY motif is mutated, phosphorylation activity on substrates like MBP (Myelin Basic Protein) is dramatically reduced to levels similar to inactive MAK mutants . The Y159 phosphorylation site is conserved across human, mouse, and rat species, suggesting its evolutionary importance in MAK function .

What are the recommended applications for Phospho-MAK (Y159) antibodies?

Phospho-MAK (Y159) antibodies are validated for several applications:

• Western Blotting (WB): The primary application with dilutions typically ranging from 1:500-1:2000

• ELISA: Can be used at approximately 1:40000 dilution

While these are the validated applications, researchers should perform optimization for their specific experimental conditions. The antibody has been tested with human cell lines including HepG2 and MCF-7, showing specific detection of MAK at approximately 70 kDa .

What are optimal storage and handling conditions?

For maximum stability and activity retention:

• Store antibody at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles by preparing aliquots before freezing

• The antibody is typically supplied in PBS buffer containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

• Some preparations may use PBS without Mg²⁺ and Ca²⁺ at pH 7.4, with 150 mM NaCl, 0.02% sodium azide, and 50% glycerol

• Working concentration is typically 1 mg/ml

How should experiments be designed to study MAK Y159 phosphorylation?

When designing experiments to study MAK Y159 phosphorylation, consider the following approach:

• Positive controls: Include samples from cells known to express phosphorylated MAK, such as HepG2 or MCF-7 cell lysates . These cell lines have been validated for MAK expression and can serve as appropriate positive controls.

• Negative controls: Consider using:

  • Cells treated with phosphatase inhibitors

  • MAK knockout/knockdown samples

  • Samples with mutated Y159 residue (Y159F substitution)

• Stimulation conditions: MAK phosphorylation can be induced through various signaling pathways. The search results indicate connections to FGFR signaling pathways, so consider FGF treatment to observe dynamic changes in phosphorylation .

• Kinase activity correlation: Plan experiments that correlate Y159 phosphorylation with MAK kinase activity using in vitro kinase assays with MBP as substrate .

What are appropriate sample preparation methods for detecting phospho-MAK (Y159)?

For optimal detection of phosphorylated MAK at Y159:

• Lysis buffer composition: Use buffer containing 50mM Tris-HCl pH7.5, 150mM NaCl, 0.5% Triton X-100, 10% glycerol, 1mM EDTA, plus phosphatase and protease inhibitors .

• Fractionation considerations: Consider nuclear and cytoplasmic fractionation when studying MAK, as its localization may change based on phosphorylation status. Commercial reagents like NE-PER Nuclear and Cytoplasmic extraction reagents can be used for this purpose .

• Immunoprecipitation protocol: For enrichment of phospho-MAK:

  • Pre-clear lysates with Protein A/G

  • Incubate with phospho-MAK antibody

  • Conjugate immune complex to protein A/G sepharose beads

  • Wash thoroughly before analysis by Western blotting

How can phospho-MAK (Y159) antibodies be used to study MAK's role in cancer?

Research indicates that MAK is overexpressed in prostate cancer cell lines and clinical specimens . To investigate this connection:

• Expression profiling: Compare phospho-MAK (Y159) levels across normal prostate tissue and progressive stages of prostate cancer to establish correlation with disease progression.

• Functional studies: Use the antibody to monitor changes in MAK phosphorylation status following treatment with anti-cancer agents or genetic manipulation of cancer-related pathways.

• Pathway analysis: Since MAK interacts with cell cycle regulation, examine the relationship between phospho-MAK (Y159) and cell cycle markers such as Cyclin B1, phospho-histone H3 Serine 10, Aurora A, and PLK1 .

• Mechanistic investigation: The antibody can be used to determine if increased phosphorylation at Y159 contributes to MAK overactivation in cancer contexts, potentially through in vitro kinase assays comparing phosphorylation levels and kinase activity.

What is the relationship between FGFR signaling and MAK Y159 phosphorylation?

The research data shows interesting connections between Fibroblast Growth Factor Receptor (FGFR) signaling and MAK/ICK:

• FGFR-MAK interaction: MAK and its homolog ICK coimmunoprecipitate with FGFR3, and ICK also interacts with FGFR1 and FGFR4, but not FGFR2 .

• Phosphorylation dynamics: When investigating this relationship, researchers should:

  • Monitor Y159 phosphorylation after FGF2 treatment

  • Assess changes in MAK kinase activity in response to FGFR activation

  • Compare phosphorylation patterns between wild-type FGFR and activating mutants like FGFR3-K650M

• Functional consequences: Evidence suggests that FGFR-mediated phosphorylation may actually inhibit ICK/MAK kinase activity by approximately 30%, despite causing accumulation of the protein . This counterintuitive relationship should be carefully examined using phospho-specific antibodies to track the dynamic regulation.

What are common issues when using phospho-MAK (Y159) antibodies in Western blots?

When working with phospho-specific antibodies, several challenges may arise:

• Specificity considerations: Ensure antibody specificity by:

  • Including phosphatase-treated controls to confirm phospho-specificity

  • Using Y159F mutants as negative controls

  • Being aware of potential cross-reactivity with related kinases like ICK, which shares homology with MAK

• Signal optimization: For optimal Western blot results:

  • Use the recommended dilution range (1:500-1:2000)

  • Consider enhanced chemiluminescent detection systems (ECL)

  • Load sufficient protein (approximately 50μg per well)

  • Use 5-20% gradient SDS-PAGE gels for optimal separation

• Blocking conditions: Use 5% non-fat milk in TBS for blocking membranes for approximately 1.5 hours at room temperature .

How can phospho-MAK (Y159) antibodies be integrated with other techniques for comprehensive analysis?

To maximize research insights:

• Complementary techniques:

  • Combine with in vitro kinase assays using MBP as substrate to correlate phosphorylation with activity

  • Pair with immunofluorescence to determine subcellular localization changes upon phosphorylation

  • Use alongside mass spectrometry to identify additional phosphorylation sites and interacting proteins

• Multi-antibody approach: Use antibodies recognizing:

  • Total MAK protein

  • Phospho-MAK (Y159)

  • Dual-phosphorylated TDY motif

  • Upstream kinases like CCRK

• Pathway integration: Since MAK relates to primary cilium function and FGFR signaling , consider studying these connections using:

  • Co-immunoprecipitation to confirm protein interactions

  • Inhibitors of FGFR to assess effects on MAK Y159 phosphorylation

  • Ciliary markers to correlate MAK phosphorylation with ciliary phenotypes

What is the potential role of MAK Y159 phosphorylation in primary cilium regulation?

Recent research indicates connections between MAK/ICK and primary cilium function:

• Functional significance: A properly functioning primary cilium is prerequisite for both normal development and aging in ciliated organisms, including humans .

• Research approach: Investigate this connection by:

  • Examining changes in MAK Y159 phosphorylation during ciliogenesis

  • Correlating phosphorylation status with ciliary length and function

  • Assessing how disruption of Y159 phosphorylation affects ciliary phenotypes

• Disease relevance: Given that ciliopathies represent an important class of genetic disorders, understanding how MAK phosphorylation contributes to ciliary regulation could have significant clinical implications.

How do multiple phosphorylation events on MAK coordinate to regulate its function?

Beyond Y159, MAK contains multiple phosphorylation sites:

• Comparative analysis: Research indicates several conserved tyrosines in ICK/MAK that can be phosphorylated by FGFR3, including Y15, Y156, Y495, and Y555 .

• Structural implications: The location of these phosphorylation sites within the protein structure has functional consequences:

  • Y15 lies within the structured kinase domain near the ATP-binding pocket

  • Y495 and Y555 are located in an unstructured regulatory region

  • Phosphorylation at Y15 may down-regulate kinase activity by interfering with ATP binding

• Investigative approach: To understand the coordination between multiple phosphorylation events:

  • Use phospho-specific antibodies to monitor different phosphorylation sites

  • Create combination mutants to assess functional interplay

  • Employ proteomics approaches to identify all relevant phosphorylation sites