CAMKK2 Antibody, FITC conjugated

What is the molecular target of CAMKK2 antibody and its biological significance?

CAMKK2 antibody targets calcium/calmodulin-dependent protein kinase kinase 2, beta (CAMKK2), a serine/threonine protein kinase with a calculated molecular weight of approximately 60 kDa (observed at 60-70 kDa in experimental conditions) . CAMKK2 plays a crucial role in calcium signaling pathways and has been implicated in various cellular processes including energy metabolism and immune cell function. Recent research has identified CAMKK2 as highly expressed within intratumoral myeloid cells in mouse models of breast cancer, suggesting its potential role as a myeloid-selective checkpoint in cancer immunology . The antibody specifically recognizes epitopes within the CAMKK2 protein, making it valuable for studying this important signaling molecule in various research contexts.

What are the optical properties and technical specifications of FITC-conjugated CAMKK2 antibody?

The FITC-conjugated CAMKK2 antibody exhibits standard fluorescein isothiocyanate optical properties with excitation/emission wavelengths of 499/515 nm and is compatible with the 488 nm laser line commonly available on flow cytometers and fluorescence microscopes . This polyclonal antibody is typically generated in rabbit hosts and shows reactivity against human CAMKK2 protein . The immunogen used for antibody production is the recombinant human CAMKK2 protein fragment spanning amino acids 5-148 , which helps ensure specific recognition of the target protein. The FITC conjugation enables direct visualization of CAMKK2 without requiring secondary antibody detection, streamlining immunofluorescence and flow cytometry protocols.

How should optimal working dilutions be determined for FITC-conjugated CAMKK2 antibody in different applications?

Determining the optimal working dilution for FITC-conjugated CAMKK2 antibody requires systematic titration in each specific application and experimental system. While manufacturer recommendations suggest that "optimal dilutions/concentrations should be determined by the end user" , a titration series starting with 1:100, 1:200, 1:400, and 1:800 dilutions is a reasonable approach for initial testing. For immunofluorescence applications, based on comparable antibodies, a dilution range of 1:200-1:800 is typically appropriate .

The titration process should include both positive controls (cells/tissues known to express CAMKK2, such as Jurkat cells, human brain tissue, HeLa cells, PC-3 cells, or RAW 264.7 cells) and negative controls (either CAMKK2-knockout samples or isotype controls). When analyzing results, look for the dilution that provides the highest signal-to-noise ratio rather than simply the strongest signal. Document the staining pattern at each dilution, as well as background levels, to establish a reproducible protocol for future experiments.

What controls should be included when using FITC-conjugated CAMKK2 antibody in flow cytometry and imaging applications?

Proper experimental controls are essential for accurate interpretation of results when using FITC-conjugated CAMKK2 antibody:

• Isotype control: Use a FITC-conjugated rabbit IgG isotype control at the same concentration as the CAMKK2 antibody to distinguish specific binding from Fc receptor-mediated or non-specific binding.

• Unstained control: Include samples without any antibody to establish autofluorescence baseline.

• Positive control: Include samples from tissues/cells known to express CAMKK2, such as PC-3 cells for immunofluorescence or Jurkat cells for flow cytometry .

• Negative control: When possible, use CAMKK2 knockout samples or cells with confirmed low expression.

• Compensation controls: For multicolor flow cytometry, include single-stained controls for each fluorophore to enable spectral compensation.

• Blocking validation: Test the specificity by pre-incubating the antibody with the immunizing peptide to confirm signal reduction.

These controls help distinguish specific from non-specific signals and provide essential reference points for data interpretation and troubleshooting.

What fixation and permeabilization protocols are recommended for optimal results with FITC-conjugated CAMKK2 antibody?

For optimal results with FITC-conjugated CAMKK2 antibody, consider the following fixation and permeabilization recommendations:

For immunohistochemistry applications of comparable CAMKK2 antibodies, antigen retrieval with TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 may serve as an alternative . This indicates that CAMKK2 epitopes may be sensitive to fixation conditions.

For immunofluorescence in cultured cells:

• Fix cells with 4% paraformaldehyde in PBS for 15-20 minutes at room temperature.

• Permeabilize with 0.1-0.5% Triton X-100 in PBS for 5-10 minutes.

• Block with 5% normal serum from the same species as the secondary antibody (if applicable) or BSA in PBS for 30-60 minutes.

• Incubate with the FITC-conjugated CAMKK2 antibody diluted in blocking buffer.

• After staining, mount with a medium containing an anti-fade agent to minimize photobleaching of FITC.

For flow cytometry:

• Fix cells with 2-4% paraformaldehyde for 10-15 minutes.

• Permeabilize with 0.1% saponin or 0.1% Triton X-100 in PBS.

• Stain in buffer containing the permeabilization agent to maintain permeabilization during antibody binding.

Since CAMKK2 has been successfully detected in various cell types including PC-3 cells , these protocols can be optimized using such cells as positive controls.

How can FITC-conjugated CAMKK2 antibody be used to study myeloid cell function in cancer immunology?

FITC-conjugated CAMKK2 antibody provides valuable tools for investigating the role of CAMKK2 in myeloid cells within the tumor microenvironment, particularly given recent findings of its relevance in cancer immunology. Research has shown that CaMKK2 is highly expressed in intratumoral myeloid cells in mouse models of breast cancer, and its inhibition may facilitate favorable reprogramming of the immune cell microenvironment .

Methodological approach for studying myeloid cell function:

• Flow cytometric analysis: Use FITC-conjugated CAMKK2 antibody in combination with myeloid lineage markers (CD11b, F4/80, CD206, MHC II) to identify and quantify specific myeloid populations expressing CAMKK2 in tumor samples. Studies have shown that tumors from Camkk2−/− mice contain different proportions of myeloid cell subsets compared to WT mice, with fewer neutrophils and MHC II− monocytes but higher percentages of macrophages and DCs .

• Sorting and functional studies: Sort CAMKK2-high versus CAMKK2-low myeloid populations using FITC signal and assess their T cell stimulatory capacity, cytokine production, and phagocytic activity. This approach can help determine whether CAMKK2 expression levels correlate with immunosuppressive functions.

• In vivo imaging: Use the FITC-conjugated antibody for intravital microscopy to track CAMKK2-expressing myeloid cells in tumor microenvironments of living organisms.

• Co-localization studies: Combine FITC-conjugated CAMKK2 antibody with markers for T cell recruitment (CXCL9, CXCL10, CXCL11) to investigate potential relationships between CAMKK2 expression and T cell chemokine production, as studies have shown upregulation of these chemokines in tumors from Camkk2−/− mice .

What strategies can be employed for multiplexing FITC-conjugated CAMKK2 antibody with other fluorophores in multi-parameter flow cytometry?

Effective multiplexing of FITC-conjugated CAMKK2 antibody with other fluorophores requires careful panel design to minimize spectral overlap and maximize signal discrimination:

• Spectral considerations: FITC (excitation/emission: 499/515 nm) has significant spectral overlap with PE, which must be addressed through proper compensation. For optimal multiplexing, combine FITC with fluorophores that have minimal spectral overlap such as APC (excitation/emission: ~650/660 nm), PE-Cy7 (excitation/emission: ~565/785 nm), or BV421 (excitation/emission: ~407/421 nm).

• Panel design strategy:

  • Assign FITC-conjugated CAMKK2 antibody to detect your protein of interest

  • Reserve brighter fluorophores (PE, APC) for targets with lower expression

  • Assign fluorophores with minimal spillover (APC, PE-Cy7) to critical markers needed for fine discrimination

• Titration for each fluorophore: Individually titrate each antibody in your panel to determine optimal signal-to-noise ratio.

• Compensation controls: Prepare single-stained controls for each fluorophore using the same cells or compensation beads. Ensure that the positive signal is in the same range as your experimental staining.

• FMO controls: Include fluorescence-minus-one controls to determine proper gating boundaries, especially for FITC which may have higher background autofluorescence.

• Experimental workflow: When combining with surface markers, perform surface staining first, then fix, permeabilize, and stain for intracellular CAMKK2 if required by your protocol.

• Buffer considerations: Use buffers that maintain FITC fluorescence intensity (pH 7.2-8.0) and include protein (0.5-1% BSA) to reduce non-specific binding .

How can researchers address high background signal when using FITC-conjugated CAMKK2 antibody?

High background signal is a common challenge when using FITC-conjugated antibodies. To address this issue with CAMKK2 antibody, consider implementing the following methodological solutions:

• Optimize blocking conditions: Increase blocking time (up to 1-2 hours) and concentration (5-10% normal serum or BSA). The buffer used for FITC-conjugated CAMKK2 antibody (0.01 M PBS, pH 7.4, with 0.03% Proclin-300 and 50% Glycerol) may be supplemented with additional blocking agents.

• Adjust antibody concentration: Excessive antibody concentration is a common cause of high background. Perform a systematic titration with dilutions ranging from 1:100 to 1:800 to identify the optimal signal-to-noise ratio.

• Increase washing steps: Add additional washing steps (at least 3-5 washes) with PBS containing 0.05-0.1% Tween-20 to remove unbound antibody.

• Reduce autofluorescence:

  • For fixed tissue: Treat with 0.1-1% sodium borohydride for 5-10 minutes before blocking

  • For cells with high autofluorescence: Consider quenching treatments or use of Sudan Black B

  • For formalin-fixed tissues: Incubate in 0.1% Sudan Black B in 70% ethanol for 20 minutes

• Evaluate fixation impact: Overfixation can increase background. If using formaldehyde, limit fixation to 10-15 minutes at room temperature.

• Use Fc receptor blocking: Pre-incubate samples with Fc block (anti-CD16/CD32) when working with immune cells to prevent non-specific Fc-mediated binding.

• Consider buffer composition: Ensure the buffer pH is optimal for FITC (pH 7.4-8.0) as lower pH can diminish fluorescence intensity.

• Use proper controls: Always compare to isotype controls and unstained samples to distinguish specific signal from background.

• Modify incubation conditions: Incubate with antibody at 4°C overnight rather than at room temperature to improve specificity of binding.

What approaches can be used to validate the specificity of FITC-conjugated CAMKK2 antibody staining?

Validating antibody specificity is critical for ensuring reliable research outcomes. For FITC-conjugated CAMKK2 antibody, implement these validation approaches:

• Genetic validation:

  • Test the antibody on CAMKK2 knockout samples (similar to the Camkk2−/− model described in research)

  • Use CAMKK2 siRNA knockdown in cell lines followed by antibody staining to confirm signal reduction

  • Test in cells with confirmed CAMKK2 overexpression to verify signal increase

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide (from amino acids 483-512 or 5-148, depending on the antibody) before staining to demonstrate signal blocking.

• Western blot correlation: Perform western blot analysis using the same antibody (unconjugated version) on the same samples to confirm that observed patterns in flow cytometry or IF match protein expression detected by western blot. Look for the expected molecular weight of CAMKK2 (60-70 kDa) .

• Multiple antibody validation: Compare staining pattern with other validated CAMKK2 antibodies targeting different epitopes, such as those targeting amino acids 1-130, 1-541, or the N-terminus .

• Positive control tissues/cells: Confirm positive staining in cells known to express CAMKK2, such as Jurkat cells, HeLa cells, PC-3 cells, or RAW 264.7 cells .

• Immunoprecipitation confirmation: Verify antibody specificity through immunoprecipitation followed by mass spectrometry to confirm the identity of the pulled-down protein.

• Co-localization studies: Perform co-staining with another validated CAMKK2 antibody or with proteins known to interact with CAMKK2 to confirm expected staining patterns.

• Cross-reactivity testing: Test the antibody against related kinases to ensure it does not cross-react with other calcium/calmodulin-dependent protein kinases.

What are the optimal storage conditions for maintaining FITC-conjugated CAMKK2 antibody performance?

Proper storage of FITC-conjugated CAMKK2 antibody is crucial for maintaining its performance over time. Based on manufacturer recommendations and best practices for fluorophore-conjugated antibodies:

Store the FITC-conjugated CAMKK2 antibody at -20°C in the dark . The antibody is typically supplied in a storage buffer containing 0.01 M PBS, pH 7.4, with 0.03% Proclin-300 and 50% Glycerol , which helps maintain stability during freeze-thaw cycles.

For long-term storage:

• Aliquot the antibody into smaller volumes upon receipt to minimize freeze-thaw cycles

• Use dark-colored tubes or wrap tubes in aluminum foil to protect from light exposure

• Store in a non-frost-free freezer at -20°C for optimal stability

• For extended storage periods (>6 months), consider storage at -80°C

For working stocks:

• Keep on ice and protected from light during experiments

• Return to 4°C storage promptly after use

• For multi-day experiments, prepare fresh dilutions daily rather than storing diluted antibody

How do freeze-thaw cycles and light exposure affect FITC-conjugated CAMKK2 antibody performance?

Freeze-thaw cycles and light exposure can significantly impact the performance of FITC-conjugated antibodies through several mechanisms:

Effects of freeze-thaw cycles:

• Protein denaturation: Repeated freezing and thawing can cause partial denaturation of the antibody protein structure, potentially reducing binding affinity and specificity

• Aggregation: Freeze-thaw cycles can promote antibody aggregation, leading to increased non-specific binding and higher background signal

• Fluorophore degradation: The FITC conjugate may detach from the antibody or degrade during freeze-thaw cycles

Effects of light exposure:

• Photobleaching: FITC is particularly susceptible to photobleaching, with estimated signal loss of 5-10% per minute under standard fluorescence microscopy illumination

• Free radical formation: Light exposure generates free radicals that can damage both the fluorophore and the antibody protein

• Decreased signal-to-noise ratio: Degraded FITC molecules may contribute to increased background while providing decreased specific signal

Methodological recommendations to minimize degradation:

• Limit exposure to light by:

  • Covering tubes with aluminum foil during all handling steps

  • Working under reduced ambient lighting conditions

  • Minimizing exposure time during fluorescence microscopy

  • Using anti-fade mounting media for microscopy preparations

• Minimize freeze-thaw cycles by:

  • Creating small single-use aliquots (10-20 μL)

  • Using a dedicated antibody freezer box to organize aliquots

  • Recording usage and freeze-thaw history for each vial

  • Discarding antibody aliquots after 5 freeze-thaw cycles

• Testing antibody performance:

  • Periodically test antibody on positive control samples

  • Document signal intensity over time to track potential degradation

  • Consider implementing standard curves with each experiment to normalize for degradation effects