Phospho-ELK1 (Thr417) Antibody

What is ELK1 and what is the significance of its phosphorylation at Thr417?

ELK1 is a member of the ETS family of transcription factors and belongs to the ternary complex factor (TCF) subfamily. It forms a ternary complex by binding to the serum response factor (SRF) and the serum response element in the promoter of the c-fos proto-oncogene, functioning as a nuclear target for the ras-raf-MAPK signaling cascade .

The phosphorylation at Thr417 is particularly significant because this modification has been shown to correlate with differentiation grade of colonic adenocarcinomas. This specific phosphorylation is reportedly mediated by cyclin-dependent kinase CDK5 . Additionally, Thr417 is one of several sites that could be phosphorylated by Cdks (alongside Thr133, Ser200, Ser202, Ser303, Ser304, Ser324, and others) . This makes Thr417 phosphorylation important both in normal cellular processes and potentially in disease states, particularly cancer progression.

How does phosphorylation of ELK1 at Thr417 differ functionally from other phosphorylation sites?

The functional differences between phosphorylation at Thr417 and other sites like Ser383 and Ser389 are significant and involve distinct signaling pathways:

Ser383 and Ser389 are preferentially phosphorylated by MAPK1 (ERK) upon mitogenic stimulation, and this phosphorylation potentiates ternary complex formation with serum response factors, SRE and SRF . These sites can also be phosphorylated by MAPK8 and/or MAKP9, leading to loss of sumoylation and restoration of transcriptional activator function .

In contrast, Thr417 phosphorylation is mediated by CDK5 and correlates with differentiation grade of colonic adenocarcinomas . This suggests that while Ser383/389 phosphorylation is more associated with mitogenic signaling through MAPK pathways, Thr417 phosphorylation might be more involved in processes related to cellular differentiation and potentially cancer progression.

Furthermore, ELK1 interacts with mitotic kinases Aurora-A, Aurora-B, Plk1, and Cdk1, with multiple phosphorylation sites potentially involved in mitotic functions . The study identified that "Elk-1 Ser304, Ser324, and Ser326 phosphorylations to be specific for mitosis," indicating different phosphorylation sites likely serve different functions throughout the cell cycle.

What are the key specifications of commercially available Phospho-ELK1 (Thr417) Antibodies?

Most commercially available Phospho-ELK1 (Thr417) Antibodies share several key specifications:

What are the validated applications for Phospho-ELK1 (Thr417) Antibody?

Based on the search results, Phospho-ELK1 (Thr417) Antibody has been validated for several applications:

How is the specificity of Phospho-ELK1 (Thr417) Antibody ensured?

The specificity of Phospho-ELK1 (Thr417) Antibody is ensured through several rigorous methodological approaches:

The antibody is produced by immunizing rabbits with synthetic phosphopeptide and KLH conjugates. The peptide sequence includes the phosphorylation site of threonine 417 (L-S-T(p)-P-V) derived from human ELK1 .

Crucially, the antibodies are purified through a two-step process: first by affinity-chromatography using epitope-specific phosphopeptide, and then non-phospho specific antibodies are removed by chromatography using non-phosphopeptide . This dual purification process ensures that the antibody specifically detects ELK1 only when phosphorylated at Thr417, without recognizing the non-phosphorylated form of the protein.

The product descriptions specifically state that the antibody "detects endogenous levels of Elk-1 protein only when phosphorylated at threonine 417" , confirming that the rigorous purification process results in an antibody with high specificity for the phosphorylated form of the protein.

How can researchers validate the specificity of Phospho-ELK1 (Thr417) Antibody in their experimental systems?

Researchers can validate the specificity of Phospho-ELK1 (Thr417) Antibody in their experimental systems through several approaches:

Positive and Negative Controls:

• Use established positive controls: Human breast carcinoma tissue for IHC and HeLa cells for ICC/IF have been verified to express phosphorylated ELK1 at Thr417 .

• Include negative controls by treating samples with phosphatase to remove phosphorylation or using tissues/cells known not to express the phosphorylated protein.

Peptide Competition Assay:

• Pre-incubate the antibody with the phosphopeptide immunogen (L-S-T(p)-P-V) before application to the sample to block specific binding.

• As a control, also pre-incubate with the non-phosphorylated peptide, which should not block specific binding.

Kinase Inhibitor Treatment:

• Treat cells with specific inhibitors of CDK5 (known to phosphorylate Thr417 ) to reduce phosphorylation and test for reduced antibody binding.

Genetic Approaches:

• Use cells with ELK1 knockdown/knockout as negative controls.

• Alternatively, use cells expressing a mutant form of ELK1 where Thr417 is replaced with alanine (T417A) to prevent phosphorylation.

Western Blot Analysis:

• Perform Western blot to confirm the antibody recognizes a protein of the expected molecular weight (approximately 45-47 kDa ).

• Include phosphatase-treated samples as controls to confirm phospho-specificity.

Immunohistochemistry (IHC):

• Dilution: 1:50-1:100

• Suggested positive control: Human breast carcinoma

• An example of successful application is shown in immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue .

Immunofluorescence (IF):

• Dilution: 1:50-1:200 , 1:100-1:200

• Suggested positive control: HeLa cells

• Methanol fixation has been demonstrated as suitable for immunofluorescence staining of HeLa cells .

Immunoprecipitation (IP):

• Recommended usage: 2-5 μg antibody per mg of lysate

ELISA:

• Dilution: 1:10000

For Immunohistochemistry (IHC):

• Use paraffin-embedded tissue sections. Successful IHC has been demonstrated with paraffin-embedded human breast carcinoma tissue .

• Standard deparaffinization, rehydration, and antigen retrieval procedures should be followed.

• Heat-induced epitope retrieval (HIER) is likely needed to unmask the phospho-epitope.

• Block endogenous peroxidase activity and non-specific binding sites.

For Immunofluorescence (IF):

• Methanol fixation has been shown to be suitable for preserving the phospho-epitope in HeLa cells .

• Alternatively, paraformaldehyde fixation followed by permeabilization with a detergent may be used.

• Blocking of non-specific binding sites is essential.

For Immunoprecipitation (IP):

• Prepare cell or tissue lysates under conditions that preserve protein phosphorylation:

  • Include phosphatase inhibitors in lysis buffers

  • Keep samples cold throughout processing

  • Use a lysis buffer compatible with the antibody's binding properties

General considerations for all applications:

• Include phosphatase inhibitors in all buffers to prevent loss of phosphorylation during sample preparation.

• Prepare samples fresh or flash-freeze and store at -80°C to preserve phosphorylation status.

• Process samples quickly to minimize potential dephosphorylation.

What is known about the interaction between ELK1 and mitotic kinases in relation to Thr417 phosphorylation?

Recent research has revealed important interactions between ELK1 and various mitotic kinases, though the specific relationship to Thr417 phosphorylation requires further investigation:

Studies have shown that "Elk-1 interacts not only with Aurora-A but also with other mitotic kinases Aurora-B, Plk1, and Cdk1, and we define the interaction domain on Elk-1 to the first N-terminal 205 amino acids" . This indicates that ELK1 has multiple interactions with key mitotic regulatory kinases.

While Thr417 has been previously reported to be phosphorylated by CDK5, research has identified it as a putative site for Cdks, which would include Cdk1, a mitotic kinase . In silico prediction of putative phosphorylation sites identified "several putative sites for Plk (Ser106, Thr108, 126, 196, and Ser326), Cdks (Thr133, Ser200, Ser202, 222, Ser303, Ser304, Ser324, 336, 353, 363, 368, Ser383, Ser389, Thr417, and 422), Aur-A and Aur-B (Ser149, Ser198, Thr199, and Ser200)" .

The study also mentions that "Elk-1 interacts with proteins such as KLF4 in PLK phosphoproteome, with proteins such as SRF, CEPT1 in Aur/PLK phosphoproteome, and with emerin (EMD) protein in both phosphoproteome datasets" , suggesting a potential role in regulating mitosis through protein-protein interactions.

How can the Phospho-ELK1 (Thr417) Antibody be used to study the role of ELK1 in cancer research?

Phospho-ELK1 (Thr417) Antibody can be a valuable tool in cancer research, particularly given the evidence linking this phosphorylation to cancer progression:

Expression Pattern Analysis:

• The phosphorylation of Elk-1 on threonine 417 has been shown to correlate with differentiation grade of colonic adenocarcinomas . The antibody can be used to examine expression and phosphorylation patterns across various cancer types and stages.

• Successful IHC staining in paraffin-embedded human breast carcinoma tissue has been demonstrated , suggesting its utility in studying breast cancer.

Prognostic Marker Investigation:

• Researchers can assess whether Thr417 phosphorylation levels correlate with patient outcomes, treatment response, or other clinical parameters through IHC analysis of tissue microarrays.

Signaling Pathway Studies:

• The antibody can help elucidate how ELK1 Thr417 phosphorylation fits into cancer-related signaling networks.

• ELK1 interacts with mitotic kinases and suggests a potential role in mitotic regulation in tumor cells .

• Researchers can study how various oncogenic signals affect Thr417 phosphorylation.

Therapy Response Monitoring:

• The antibody can monitor changes in ELK1 phosphorylation in response to cancer therapies, particularly those targeting CDK5 or related pathways.

Cellular Localization:

• Using IF (as demonstrated with HeLa cells ), researchers can study the subcellular localization of phosphorylated ELK1 in cancer cells and how it might change with disease progression or in response to treatments.

How can Phospho-ELK1 (Thr417) Antibody be combined with other markers to study signaling pathways?

Phospho-ELK1 (Thr417) Antibody can be effectively combined with other markers to provide comprehensive insights into signaling pathways:

Multiplex Immunostaining/Immunofluorescence:

• Combine with antibodies against other components of relevant signaling pathways including:

  • CDK5 and its activators (p35/p25), as CDK5 phosphorylates ELK1 at Thr417

  • Mitotic kinases (Aurora-A, Aurora-B, Plk1, Cdk1) that interact with ELK1

  • Other phosphorylated forms of ELK1 (e.g., at Ser383/389) to compare different activation states

  • Downstream targets of ELK1-mediated transcription

Co-immunoprecipitation Studies:

• Use the antibody for immunoprecipitation followed by Western blotting for interacting proteins.

• This can identify protein complexes associated with phosphorylated ELK1.

• Potential interaction partners include KLF4, SRF, CEPT1, and emerin (EMD) .

Pathway Inhibitor Studies:

• Combine with treatments using specific inhibitors of relevant kinases (CDK5, MAPKs, Aurora kinases, Plk1) to dissect pathway hierarchies.

• Monitor how inhibition of different pathway components affects Thr417 phosphorylation.

Cell Cycle Analysis:

• Given the potential role in mitosis , combine with cell cycle markers (e.g., phospho-histone H3, cyclin B1) to study the relationship between ELK1 phosphorylation and cell cycle progression.

Chromatin Immunoprecipitation (ChIP) Assays:

• Use the antibody for ChIP to identify genomic regions bound by phosphorylated ELK1.

• This can reveal how Thr417 phosphorylation affects ELK1's DNA binding and transcriptional regulatory functions.

How can researchers troubleshoot non-specific binding when using Phospho-ELK1 (Thr417) Antibody?

When encountering non-specific binding issues with Phospho-ELK1 (Thr417) Antibody, several troubleshooting approaches can be implemented:

Optimize Antibody Dilution:

• The recommended dilutions vary depending on the application: 1:50-1:100 for IHC , 1:50-1:200 for IF , and 1:10000 for ELISA .

• If experiencing non-specific binding, try more diluted antibody concentrations.

Improve Blocking:

• Increase blocking time or try different blocking agents (e.g., BSA, normal serum, commercial blocking solutions).

• Some antibody formulations already contain 0.5% BSA , but additional blocking may be necessary.

Increase Washing Stringency:

• Use more wash steps or increase the duration of washing.

• Consider adding a small amount of detergent to wash buffers.

Validate Antibody Specificity:

• Perform a peptide competition assay using the phosphopeptide immunogen (L-S-T(p)-P-V) to confirm whether the observed signal is specific.

• The antibody production process removes non-phospho specific antibodies , so theoretically, the antibody should be highly specific.

Use Additional Controls:

• Include a phosphatase-treated sample to confirm phospho-specificity.

• Use tissues or cells known not to express ELK1 as negative controls.

Modify Fixation or Antigen Retrieval:

• Try different fixation methods or optimize antigen retrieval conditions.

• Methanol fixation has shown successful results with HeLa cells for IF .

What controls should be included when using Phospho-ELK1 (Thr417) Antibody?

When using Phospho-ELK1 (Thr417) Antibody, researchers should include several types of controls:

Positive Controls:

• Use samples known to express phosphorylated ELK1 at Thr417. Human breast carcinoma is suggested for IHC and HeLa cells for ICC/IF .

• Include samples treated with agents known to induce this phosphorylation.

Negative Controls:

• Omit primary antibody but include all other reagents to check for non-specific binding of detection systems.

• Use samples known not to express ELK1 or the phosphorylated form.

• Include samples treated with phosphatases to remove phosphorylation.

Specificity Controls:

• Perform peptide competition assay: Pre-incubate the antibody with the phosphopeptide immunogen (L-S-T(p)-P-V ) to block specific binding.

• Also pre-incubate with the non-phosphorylated peptide, which should not block specific binding if the antibody is truly phospho-specific.

Kinase Inhibition Controls:

• Treat samples with inhibitors of kinases known to phosphorylate Thr417 (such as CDK5 inhibitors ) to demonstrate reduced signal.

Technical Controls:

• Include isotype control antibodies (rabbit IgG at the same concentration, as the Phospho-ELK1 (Thr417) Antibody is a rabbit polyclonal ).

• Use different antibody dilutions to establish optimal signal-to-noise ratio.

Including these controls will help ensure that the observed signals truly represent phosphorylated ELK1 at Thr417 and not artifacts or non-specific binding.