MAPK1 Antibody

What is MAPK1 and what cellular functions does it regulate?

MAPK1 (also known as ERK2) is a serine/threonine kinase that functions as an essential component of the MAP kinase signal transduction pathway. MAPK1/ERK2 and MAPK3/ERK1 are two MAPKs that play crucial roles in the MAPK/ERK cascade. This pathway mediates diverse biological functions including cell growth, adhesion, survival, and differentiation through the regulation of transcription, translation, and cytoskeletal rearrangements. MAPK1 is involved in the initiation and regulation of meiosis, mitosis, and postmitotic functions in differentiated cells by phosphorylating numerous transcription factors. Approximately 160 substrates have been identified for ERKs, including nuclear transcription factors regulating gene expression, cytosolic proteins controlling translation, and factors involved in mitosis and apoptosis .

In which tissues and cellular locations is MAPK1 typically expressed?

MAPK1 exhibits a broad tissue distribution pattern. According to research data, it is expressed in brain, heart, placenta, pancreas, and skeletal muscle . At the subcellular level, MAPK1 can be found in multiple locations including:

• Cytoplasm and cytoskeleton

• Nucleus

• Centrosome

• Spindle during prometaphase and metaphase

Its localization is dynamically regulated. For instance, phosphorylation at Ser-246 and Ser-248, as well as autophosphorylation at Thr-190, promote nuclear localization. Conversely, PEA15-binding and phosphorylated DAPK1 promote cytoplasmic retention of MAPK1 .

What are the recommended applications for MAPK1 antibodies?

MAPK1 antibodies have been validated for several research applications with specific dilution recommendations:

The appropriate application should be selected based on your experimental objectives. Expression analysis in cell lines like HeLa has confirmed robust MAPK1 expression, making these suitable positive controls .

How should MAPK1 antibodies be stored and reconstituted for optimal performance?

For short-term storage (up to 2 weeks), maintain MAPK1 antibodies refrigerated at 2-8°C. For long-term storage, aliquot the antibody and store at -20°C to prevent freeze-thaw cycles that can degrade antibody quality. Most commercial MAPK1 antibodies are supplied in PBS with 0.09% (W/V) sodium azide. When reconstituting lyophilized antibodies, use the recommended buffer (typically PBS) and prepare small aliquots to minimize freeze-thaw cycles. Actual concentration may vary between lots, but is typically approximately 0.5mg/ml .

How do MAPK1 antibodies compare with antibodies targeting other MAPK pathway components in multiplex assays?

When designing multiplex assays involving multiple MAPK pathway components, it's important to understand the relationship between various antibodies. Publication data indicates significant research overlap between MAPK14 (p38 MAPK) and MAPK1, with over 37 publications utilizing both antibodies. Similarly, MAPK3 antibodies have been used alongside MAPK14 in more than 30 publications, suggesting compatible assay conditions. Other frequently co-investigated targets include MAPK8 (19+ publications), MAPKAPK2 (17+ publications), MAP2K6 (15+ publications), and MAP2K3 (14+ publications) .

For multiplex detection, consider the following methodological approach:

• Validate each antibody individually first

• Ensure antibodies are raised in different host species or use isotype-specific secondary antibodies

• Test for cross-reactivity between secondary antibodies

• Optimize blocking conditions to minimize background

• Consider sequential rather than simultaneous detection for closely related targets

What methodological considerations should be addressed when using MAPK1 antibodies for studying phosphorylation-dependent signaling?

When investigating MAPK1 phosphorylation states, several critical methodological considerations must be addressed:

• Sample preparation: Rapid sample processing is essential as phosphorylation states are labile. Use phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate) in lysis buffers.

• Antibody selection: Use phospho-specific antibodies that recognize specific phosphorylation sites (such as Thr-185/Tyr-187) alongside total MAPK1 antibodies.

• Controls: Include both positive controls (stimulated cells known to activate MAPK1) and negative controls (cells treated with MAPK pathway inhibitors).

• Normalization strategy: Always normalize phospho-MAPK1 signal to total MAPK1 levels to account for variations in total protein expression.

• Temporal dynamics: Design time-course experiments to capture the transient nature of phosphorylation events.

Research has demonstrated that MAPK1 plays roles in signaling cascades initiated by activated KIT and KITLG/SCF, where phosphorylation state is critical for function . Additionally, understanding the interaction between MAPK1 and other pathway components like DNA-PKcs is essential, as these interactions can regulate activity independently of phosphorylation status .

How can researchers distinguish between MAPK1 (ERK2) and MAPK3 (ERK1) when using antibodies in experimental systems?

Distinguishing between MAPK1 (ERK2) and MAPK3 (ERK1) is crucial due to their structural similarity (84% amino acid identity) but potentially distinct functions. The following methodological approach is recommended:

• Antibody selection: Use antibodies raised against unique epitopes in the central region (amino acids 154-183) of MAPK1, which shows greater sequence divergence from MAPK3 .

• Molecular weight differentiation: In Western blotting, MAPK1/ERK2 runs at approximately 42 kDa while MAPK3/ERK1 runs at approximately 44 kDa.

• Knockout/knockdown validation: Validate antibody specificity using MAPK1-specific siRNA knockdown or CRISPR knockout cells to confirm band identity.

• Peptide competition: Perform peptide competition assays using the immunizing peptide to confirm antibody specificity.

• Isoform-specific substrates: Some substrates show preference for MAPK1 over MAPK3; these can be used as functional readouts.

When reporting results, clearly specify which isoform was detected and include molecular weight markers to substantiate claims of isoform specificity.

What is the role of MAPK1 in cancer progression, and how can MAPK1 antibodies be utilized in cancer research?

MAPK1 plays significant roles in cancer progression through its involvement in cell proliferation, survival, and metastasis. MAPK1 antibodies serve as valuable tools in investigating these processes:

• Expression analysis: MAPK1 antibodies can be used to evaluate expression levels across different tumor types and stages using IHC-P (1:50-1:100 dilution) .

• Activation status: Phospho-specific antibodies can determine MAPK pathway activation status, which has implications for therapy selection.

• Therapeutic response markers: Changes in MAPK1 expression or phosphorylation can serve as biomarkers for response to targeted therapies.

• Combination therapy research: MAPK1 antibodies can assess pathway modulation in combination therapy approaches. For example, research has shown that MAPK pathway inhibitors sensitize BRAF-mutant melanoma to antibody-drug conjugates targeting GPNMB. In these studies, MAPK inhibitor treatment induces MITF-dependent expression of melanosomal differentiation genes, including GPNMB, rendering tumor cells susceptible to targeted therapy .

• Immunotherapy resistance mechanisms: MAPK1 antibodies can help investigate resistance mechanisms to immunotherapies. Research has demonstrated that the p38 MAPK pathway influences the efficacy of B7-H1 (PD-L1) antibodies in cancer immunotherapy. Specifically, certain B7-H1 antibodies can activate p38 MAPK, leading to deletion of B7-H1+ tumor-reactive CD8+ T cells and diminished antitumor activity .

What strategies can be employed to troubleshoot inconsistent or negative results when using MAPK1 antibodies?

When encountering inconsistent or negative results with MAPK1 antibodies, implement the following troubleshooting strategies:

• Antibody validation: Confirm antibody functionality using positive control samples. For instance, HeLa cells are confirmed to express MAPK1 and serve as appropriate positive controls .

• Epitope accessibility: Consider that some epitopes may be masked by protein-protein interactions or post-translational modifications. Try multiple antibodies targeting different regions of MAPK1.

• Sample preparation optimization:

  • For Western blotting: Optimize lysis conditions and denaturants

  • For IHC: Test different antigen retrieval methods (heat-induced vs. enzymatic)

  • For Flow cytometry: Ensure proper permeabilization for intracellular targets

• Signal enhancement techniques: For weak signals, consider:

  • Signal amplification systems (e.g., tyramide signal amplification)

  • More sensitive detection methods (e.g., chemiluminescence vs. colorimetric)

  • Extended antibody incubation times at lower temperatures (4°C overnight)

• Methodological analysis: Some antibodies may work in certain applications but not others. Affinity measurements using techniques like Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry can provide objective assessment of antibody-antigen interactions. For instance, some MAPK1 antibodies show "No Binding" results in these assays despite working in other applications like immunohistochemistry .

How should experiments be designed to study MAPK1 interaction with its substrates using antibody-based approaches?

Designing experiments to study MAPK1-substrate interactions requires careful planning:

• Co-immunoprecipitation (Co-IP) protocol optimization:

  • Use mild lysis conditions to preserve protein-protein interactions

  • Consider crosslinking to stabilize transient interactions

  • Compare results using antibodies against both MAPK1 and the substrate of interest

• Proximity ligation assay (PLA) implementation:

  • Provides spatial information about protein interactions in situ

  • Requires antibodies from different species against MAPK1 and its substrate

  • Includes appropriate controls (single antibody controls)

• Kinase assay design:

  • Use immunoprecipitated MAPK1 to assess substrate phosphorylation

  • Include both active (phosphorylated) and inactive MAPK1 conditions

  • Verify substrate specificity using MAPK inhibitors

• Substrate specificity analysis:

  • Focus on known MAPK1 substrates including transcription factors (ATF2, BCL6, ELK1, ERF, FOS), cytoskeletal elements (CANX, CTTN, MAP2, MAPT), and regulators of apoptosis (BAD, CASP9, MCL1)

  • Consider the subcellular localization of both MAPK1 and potential substrates

Data from research indicates that MAPK1 has approximately 160 substrates with diverse cellular functions, requiring careful experimental design to distinguish direct from indirect effects .

What controls should be included when validating MAPK1 antibody specificity for different research applications?

Rigorous validation of MAPK1 antibodies requires comprehensive controls:

• Positive and negative tissue/cell controls:

  • Positive: Brain, heart, placenta, pancreas, and skeletal muscle tissues show high MAPK1 expression

  • Positive cell line: HeLa cells have confirmed MAPK1 expression

  • Negative: Use tissues or cell lines with minimal MAPK1 expression

• Genetic controls:

  • MAPK1 knockdown (siRNA) or knockout (CRISPR-Cas9) cells

  • Overexpression systems with tagged MAPK1 constructs

• Peptide competition:

  • Preincubate antibody with immunizing peptide (e.g., synthetic peptide from amino acids 154-183 of human MAPK1)

  • Include gradient of peptide concentrations to demonstrate specificity

• Cross-reactivity assessment:

  • Test against closely related proteins, particularly MAPK3/ERK1

  • Evaluate antibody performance across species boundaries using sequence homology data

  • Common cross-reactivity observed in Bovine, Human, Mouse, Rat, and Xenopus samples

• Application-specific controls:

  • For IHC: Include isotype control antibodies, secondary-only controls

  • For WB: Include molecular weight markers, loading controls

  • For IP: Include IgG control immunoprecipitations

  • For FC: Include fluorescence-minus-one (FMO) controls

How can MAPK1 antibodies be utilized to investigate therapeutic resistance mechanisms in cancer?

MAPK1 antibodies serve as critical tools for investigating therapeutic resistance:

• Monitoring pathway reactivation:

  • Use phospho-specific MAPK1 antibodies to detect reactivation of MAPK signaling after initial response to targeted therapies

  • Apply in time-course experiments to determine when pathway reactivation occurs

• Identifying bypass mechanisms:

  • Compare MAPK1 activation with parallel pathways (PI3K/AKT, JAK/STAT) to identify compensatory signaling

  • Use multiplex immunofluorescence to assess co-activation patterns in single cells

• Evaluating combination therapy strategies:

  • Research indicates that MAPK pathway inhibitors can sensitize BRAF-mutant melanoma to antibody-drug conjugates targeting GPNMB

  • BRAF and MEK inhibitor treatment induces MITF-dependent expression of melanosomal differentiation genes, including GPNMB, making tumor cells susceptible to targeted therapy with CDX-011

• Investigating post-transcriptional modifications:

  • Beyond phosphorylation, investigate other modifications (ubiquitination, SUMOylation) using appropriate antibodies

  • Correlate modifications with resistance phenotypes

• Tumor microenvironment interactions:

  • Research has shown that p38 MAPK activation influences immune checkpoint therapy outcomes

  • B7-H1 (PD-L1) antibodies capable of activating p38 MAPK lose antitumor activity by deleting B7-H1+ tumor-reactive CD8+ T cells

What methodological approaches can be used to study MAPK1 pathway dynamics in response to therapeutic interventions?

Investigating MAPK1 pathway dynamics requires sophisticated methodological approaches:

• Time-resolved analysis protocols:

  • Design sampling timepoints based on known MAPK activation kinetics (early phase: 5-30 minutes; sustained phase: 1-24 hours)

  • Use phospho-specific antibodies targeting different sites (e.g., Thr-185/Tyr-187)

  • Implement rapid fixation protocols to "freeze" signaling states

• Single-cell analysis approaches:

  • Use phospho-flow cytometry with MAPK1 antibodies to assess heterogeneity in response

  • Combine with markers of cell cycle, apoptosis to correlate pathway activity with cellular outcomes

  • Consider mass cytometry (CyTOF) for higher-dimensional analysis

• Live-cell imaging considerations:

  • Use FRET-based biosensors for MAPK activity rather than direct antibody application

  • Correlate imaging data with fixed-cell antibody-based approaches for validation

• Drug-response profiling designs:

  • Implement concentration gradients and time-course experiments

  • Include clinically relevant drug concentrations

  • Assess pathway reactivation patterns during drug holidays

• Analysis of clinical samples:

  • Design protocols for serial biopsies before, during, and after therapy

  • Use immunohistochemistry with MAPK1 antibodies to assess pathway activity in patient samples

  • Integrate with serum biomarker analysis

  • This approach has been successfully utilized in melanoma patients receiving MAPK pathway inhibitors

How can MAPK1 antibodies be utilized in studies investigating protein-protein interactions and signaling complexes?

MAPK1 antibodies offer valuable tools for investigating complex formation and protein interactions:

• Co-immunoprecipitation (Co-IP) optimization:

  • Use antibodies targeting different epitopes to avoid disrupting interaction surfaces

  • Consider native vs. crosslinked conditions to capture transient interactions

  • Include appropriate controls (IgG, reverse Co-IP)

• Proximity-based detection methods:

  • Proximity Ligation Assay (PLA) can visualize MAPK1 interactions in situ

  • BRET/FRET approaches complement antibody-based methods for live-cell studies

• Protein complex isolation:

  • Use sequential immunoprecipitation to isolate specific complexes

  • Combine with mass spectrometry for unbiased interactome analysis

  • Research has identified DNA-PKcs as a novel intracellular partner of B7-H1 in the cytoplasm of activated CD8+ T cells, with implications for p38 MAPK regulation

• Scaffolding protein analysis:

  • Investigate interactions with known MAPK scaffolds (KSR, MP1, IQGAP)

  • Use domain-specific antibodies to map interaction regions

• Subcellular fractionation approaches:

  • Combine with immunoblotting to track MAPK1 localization and complex formation

  • Include markers for specific compartments (nuclear, cytoplasmic, cytoskeletal)

  • Consider the dynamic localization of MAPK1 across cytoplasm, cytoskeleton, spindle, nucleus, and centrosome

What considerations are important when using MAPK1 antibodies in studies of post-translational modifications beyond phosphorylation?

While phosphorylation is the most studied MAPK1 modification, other post-translational modifications (PTMs) significantly impact function:

• PTM-specific antibody selection:

  • Beyond phosphorylation, consider antibodies against acetylation, ubiquitination, and SUMOylation of MAPK1

  • Validate PTM specificity using appropriate controls (phosphatase treatment, deubiquitinating enzymes)

• Enrichment strategies:

  • Use PTM enrichment methods prior to antibody-based detection for low-abundance modifications

  • Consider sequential immunoprecipitation (first with MAPK1 antibody, then with PTM-specific antibody)

• Modification site mapping:

  • Use site-specific antibodies when available

  • Complement with mass spectrometry approaches for site identification

  • Compare PTM patterns with those of known MAPK1 functions and localizations

• Cross-talk analysis:

  • Investigate relationships between phosphorylation and other PTMs

  • Design experiments to determine sequential modification patterns

  • Consider the impact of phosphorylation at sites like Ser-246 and Ser-248 on nuclear localization

• Functional validation:

  • Correlate detected modifications with MAPK1 activity, localization, and protein-protein interactions

  • Use mutational approaches (site-directed mutagenesis) to validate functional significance