CAMK1D Antibody

What is CAMK1D and why is it important to study?

CAMK1D (Calcium/calmodulin-dependent protein kinase type 1D) is a serine/threonine kinase that operates in the calcium-triggered CaMKK-CaMK1 signaling cascade. Upon calcium influx, it activates CREB-dependent gene transcription, regulates calcium-mediated granulocyte function and respiratory burst, and promotes basal dendritic growth of hippocampal neurons . In neutrophil cells, it is required for cytokine-induced proliferative responses and activation of the respiratory burst . Recent research has implicated CAMK1D in cancer progression, particularly in breast cancer where it has been associated with epithelial-mesenchymal transition (EMT) . It has also been identified as a key modulator of tumor-intrinsic immune resistance .

What types of CAMK1D antibodies are available for research applications?

Based on the search results, various types of CAMK1D antibodies are available for research purposes:

These antibodies recognize different epitopes of CAMK1D, providing researchers with options based on their specific experimental requirements .

What are the most common applications for CAMK1D antibodies?

CAMK1D antibodies are primarily used in the following applications:

• Western Blot (WB): For detecting and quantifying CAMK1D protein levels in cell or tissue lysates. Typically used at dilutions ranging from 1:500 to 1:2000 .

• Immunohistochemistry (IHC): For visualizing CAMK1D expression in paraffin-embedded tissue sections. Recommended dilutions are often between 1:20 to 1:50 .

• Immunofluorescence (IF)/Immunocytochemistry (ICC): For cellular localization studies. Typically used at dilutions of 1:50 to 1:200 .

• ELISA: For quantitative measurement of CAMK1D. Usually employed at dilutions around 1:4000 .

How should I optimize Western blot conditions for CAMK1D detection?

When performing Western blot for CAMK1D detection:

• Sample preparation: Use RIPA buffer for efficient protein extraction from tissues or cells .

• Protein loading: Load approximately 35 μg of total protein per lane for optimal detection .

• Antibody concentration: For most CAMK1D antibodies, a dilution range of 0.5-2 μg/mL works well .

• Incubation time: Primary antibody incubation of 1 hour at room temperature or overnight at 4°C is recommended .

• Detection method: Chemiluminescence provides sensitive detection of CAMK1D .

• Expected molecular weight: Look for bands around 42.9-43 kDa, which corresponds to the calculated molecular weight of CAMK1D .

For validation of specificity, knockout samples can be used as negative controls, as demonstrated with ab172618 which has been shown to specifically react with CAMK1D when CAMK1D knockout samples were used .

What are the critical parameters for immunohistochemical staining with CAMK1D antibodies?

For optimal immunohistochemical staining of CAMK1D:

• Tissue preparation: Use paraffin-embedded tissue sections, properly fixed to preserve antigenicity .

• Antigen retrieval: This step is crucial for unmasking epitopes that may be cross-linked during fixation.

• Antibody dilution: Start with a dilution range of 1:20 to 1:50 for most CAMK1D antibodies .

• Controls: Include positive controls (tissues known to express CAMK1D) and negative controls (primary antibody omitted) in each experiment.

• Detection system: Use appropriate secondary antibodies and visualization methods compatible with your primary antibody.

Immunohistochemical analysis has revealed elevated CAMK1D expression in invasive carcinomas compared to carcinoma in situ, suggesting its utility as a biomarker for cancer progression .

How can I determine the optimal concentration of CAMK1D antibody for kinase binding assays?

For LanthaScreen® Eu Kinase Binding Assays with CAMK1D:

• Initial setup: Follow a two-step process - first optimize the tracer concentration, then perform the kinase inhibitor affinity (IC50) measurements .

• Recommended conditions:

  • 5 nM kinase

  • 2 nM Eu-Streptavidin

  • 2 nM Biotin Anti-His Tag Antibody

  • 30 nM Kinase Tracer 236

  • 1X Kinase Buffer A

• Protocol sequence:

  • Add 5 µL of test compound

  • Add 5 µL of kinase/antibody mixture

  • Add 5 µL of tracer

  • Incubate for 1 hour at room temperature before reading the plate

• Data analysis: Calculate the emission ratio by dividing the acceptor/tracer emission (665 nm) by the antibody/donor emission (615 nm) .

How can CAMK1D antibodies be used to investigate its role in cancer immune resistance?

Recent research has identified CAMK1D as a key modulator of tumor-intrinsic immune resistance . When investigating this role:

• Co-expression analysis: Use CAMK1D antibodies alongside PD-L1 antibodies to study their co-expression in anti–PD-L1/PD-1 treatment–refractory cancer types .

• Activation mechanism: CAMK1D is activated by CTL through Fas-receptor stimulation, which leads to CAMK1D binding to and phosphorylating caspase-3, -6, and -7, inhibiting their activation and function .

• Inhibition studies: Use CAMK1D antibodies to validate the effects of pharmacologic inhibition of CAMK1D, which has been shown to restore sensitivity toward Fas-ligand treatment in multiple myeloma and uveal melanoma cells in vitro .

• In vivo models: CAMK1D has been shown to mediate immune resistance of murine colorectal cancer cells in vivo, providing a model system for studying therapeutic interventions .

This research pathway is particularly relevant for developing new strategies to overcome resistance to immune checkpoint blockade therapies.

What is the relationship between CAMK1D and epithelial-mesenchymal transition (EMT) in cancer?

CAMK1D has been implicated in epithelial-mesenchymal transition, a process critical for cancer invasion and metastasis :

• Amplification detection: Genomic profiling using CAMK1D antibodies can help identify amplification of CAMK1D in basal-like breast tumors, which has been associated with poor prognosis .

• Expression analysis: By immunohistochemistry, CAMK1D exhibits elevated expression in invasive carcinomas compared to carcinoma in situ .

• Functional studies: When overexpressed in non-tumorigenic breast epithelial cells (such as MCF10A), CAMK1D leads to:

  • Increased cell proliferation

  • Loss of cell-cell adhesions (decreased E-cadherin expression)

  • Increased vimentin expression (a mesenchymal marker)

  • Enhanced cell migration and invasion

• Pathway analysis: Gene set enrichment analysis (GSEA) of CAMK1D-expressing cells reveals enrichment of EMT-relevant pathways, cell proliferation pathways, and CREB regulatory motifs .

How can CAMK1D-specific inhibitors be evaluated in neuronal models?

When studying CAMK1D inhibitors in neuronal models:

• Cell viability assays: Assess the effects of CAMK1D inhibitors (e.g., CS587) on neuronal viability at various concentrations (1 nM to 1 µM) .

• Dendritic growth analysis: Since CAMK1D promotes basal dendritic growth of hippocampal neurons, measure dendritic arborization in the presence and absence of inhibitors .

• CREB activation: Monitor CREB phosphorylation status using phospho-specific antibodies, as CAMK1D activates the transcription factor CREB1 in hippocampal neuron nuclei .

• Calcium imaging: Combine with calcium imaging techniques to correlate calcium influx with CAMK1D activity and inhibition effects.

Research has shown that CAMK1D inhibitors can be used in media concentrations up to 1 µM without detrimental effects on neuronal cell viability .

How do I validate the specificity of a CAMK1D antibody?

To ensure antibody specificity:

• Knockout/knockdown controls: Use CAMK1D knockout or knockdown samples as negative controls. For example, ab172618 was validated using CAMK1D knockout samples .

• Peptide competition: Pre-incubate the antibody with the immunogen peptide before application to verify that binding is specific.

• Multiple antibodies: Use different antibodies targeting distinct epitopes of CAMK1D and compare results.

• Cross-reactivity testing: Check reactivity against related proteins, particularly other CAMK family members.

• Western blot analysis: Verify that the detected band corresponds to the expected molecular weight of CAMK1D (approximately 43 kDa) .

What factors influence reproducibility in CAMK1D antibody-based experiments?

Several factors can affect experimental reproducibility:

• Antibody storage: Store antibodies according to manufacturer recommendations to maintain activity. Most CAMK1D antibodies should be stored at -20°C .

• Sample preparation: Consistent protocols for sample collection, fixation, and processing are essential.

• Antibody lot-to-lot variation: When possible, use the same lot of antibody for related experiments or validate new lots against previous results.

• Experimental conditions: Standardize incubation times, temperatures, and buffer compositions.

• Controls: Always include appropriate positive and negative controls.

For instance, in one trial with CS587 (a CAMK1D inhibitor), increased viability of neurons was observed at concentrations of 1 nM and 10 nM, but this effect was not reproducible in subsequent experiments , highlighting the importance of replication.

What are the considerations for selecting the appropriate CAMK1D antibody for different experimental purposes?

When selecting a CAMK1D antibody:

Additionally, consider:

• The epitope region recognized by the antibody (e.g., N-terminal, C-terminal, or internal regions)

• Host species (to avoid cross-reactivity in multi-color applications)

• Validation data provided by the manufacturer

• Published literature using the specific antibody