CSNK1G3 Antibody, FITC conjugated

What is CSNK1G3 and what cellular functions does it regulate?

CSNK1G3 is a serine/threonine protein kinase belonging to the casein kinase 1 family, specifically the gamma subfamily. It is also known as casein kinase I gamma 3, CKI-gamma 3, or KC1G3_HUMAN. The protein has a molecular weight of approximately 51 kDa and is encoded by the CSNK1G3 gene . Unlike its family member CSNK1G2, CSNK1G3 does not appear to suppress RIPK3 kinase activity, suggesting distinct functional roles between these closely related kinases . CSNK1G3 is involved in various cellular processes including signal transduction pathways, DNA repair, and cellular differentiation, though its precise roles continue to be investigated.

What are the typical applications for a FITC-conjugated CSNK1G3 antibody?

A FITC-conjugated CSNK1G3 antibody is primarily utilized in fluorescence-based detection methods. While the unconjugated antibody is suitable for Western blot (WB) and immunohistochemistry (IHC) , the FITC-conjugated version extends its applications to:

• Flow cytometry for quantitative analysis of CSNK1G3 expression in cell populations

• Immunofluorescence microscopy for subcellular localization studies

• Fluorescence-based immunoassays for protein detection

• Live-cell imaging (for membrane-associated targets)

The FITC conjugation eliminates the need for secondary antibody incubation steps, which can reduce background signal and simplify experimental workflows in fluorescence-based applications .

What species reactivity can be expected with the CSNK1G3 antibody?

The CSNK1G3 antibody has confirmed reactivity with human samples and is also reactive with mouse and rat specimens . Computational predictive modeling suggests potential cross-reactivity with samples from pig, bovine, horse, sheep, rabbit, dog, and Xenopus species, though these interactions should be experimentally validated before use in critical experiments . When studying model organisms, it is advisable to perform preliminary validation experiments to confirm antibody specificity, especially when working with species not explicitly listed in the manufacturer's specifications.

What are the optimal storage conditions for maintaining FITC-conjugated CSNK1G3 antibody activity?

To maintain optimal activity of the FITC-conjugated CSNK1G3 antibody, adhere to the following storage guidelines:

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

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

• Protect from light due to the photosensitivity of the FITC fluorophore

• Avoid repeated freeze-thaw cycles as they can degrade both the antibody and the fluorophore

• The antibody is typically supplied in a buffer containing 0.01 M PBS (pH 7.4), 0.03% Proclin-300, and 50% glycerol, which helps maintain stability during storage

When handling the antibody, minimize exposure to light and keep on ice during experimental procedures to preserve the fluorescence intensity.

How can I optimize immunofluorescence protocols using FITC-conjugated CSNK1G3 antibody to minimize autofluorescence and background?

Optimizing immunofluorescence protocols with FITC-conjugated CSNK1G3 antibody requires careful consideration of several parameters:

• Fixation method selection: Compare paraformaldehyde, methanol, and acetone fixation to determine which best preserves CSNK1G3 epitopes while minimizing autofluorescence. Typically, 4% paraformaldehyde for 15-20 minutes preserves most epitopes.

• Autofluorescence reduction:

  • Include a 10-minute treatment with 50 mM NH₄Cl in PBS after fixation to quench aldehyde-induced autofluorescence

  • For tissues with high lipofuscin content, treat with 0.1% Sudan Black B in 70% ethanol for 20 minutes

  • Consider using specialized commercial reagents designed to reduce autofluorescence

• Blocking optimization: Test different blocking solutions (5% normal serum, 3% BSA, commercial blocking reagents) to determine which most effectively reduces non-specific binding.

• Antibody titration: Perform a dilution series (typically starting at 1:50, 1:100, 1:200, 1:500) to identify the optimal antibody concentration that maximizes specific signal while minimizing background .

• Buffer composition: Include 0.1% Triton X-100 for intracellular targets, but reduce to 0.01% or omit for membrane proteins.

• Counterstaining selection: Choose counterstains that minimize spectral overlap with FITC (emission peak ~520 nm). DAPI (blue) or far-red nuclear stains are preferred over propidium iodide (which has some spectral overlap with FITC).

• Negative controls: Include a matching isotype control antibody (rabbit IgG-FITC) to assess non-specific binding of the primary antibody.

What experimental approaches should I use to validate CSNK1G3 antibody specificity for my particular application?

Thorough validation of the CSNK1G3 antibody specificity is critical for generating reliable data. Implement the following approaches:

• Genetic validation:

  • CRISPR/Cas9 knockout or siRNA knockdown of CSNK1G3 in your cell system, followed by antibody testing to confirm signal reduction

  • Overexpression of tagged CSNK1G3 to verify co-localization with antibody signal

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide (if available) before application to samples; specific signal should be significantly reduced.

• Western blot correlation: For fluorescence applications, correlate observations with Western blot results using the same antibody to confirm target molecular weight (expected 51 kDa) .

• Multiple antibody verification: Compare results with a second antibody targeting a different epitope of CSNK1G3.

• Phosphorylation sensitivity assessment: If studying phosphorylation-dependent events, determine if the antibody recognition is affected by phosphorylation status by comparing samples treated with phosphatase inhibitors versus phosphatase-treated samples.

• Cross-reactivity testing: Test reactivity against purified CSNK1G1 and CSNK1G2 proteins to ensure specificity within the casein kinase family, particularly important given the structural similarities between these family members .

• Mass spectrometry validation: For definitive validation, perform immunoprecipitation followed by mass spectrometry to confirm target identity.

How can I design multiplexed experiments to study CSNK1G3 interactions with other proteins using the FITC-conjugated antibody?

Designing effective multiplexed experiments to study CSNK1G3 interactions requires careful planning:

• Fluorophore selection for multiplex imaging:

  • Pair FITC-conjugated CSNK1G3 antibody (green) with far-red (e.g., Alexa Fluor 647) or red (e.g., Alexa Fluor 594) conjugated antibodies against potential interaction partners

  • Avoid PE or other yellow-orange fluorophores that may have spectral overlap with FITC

• Sequential immunostaining protocol:

  • For co-staining with other rabbit antibodies, perform sequential staining with a direct conjugate (FITC-CSNK1G3) first

  • Block with excess rabbit IgG between steps

  • Follow with unconjugated primary and species-specific secondary antibody combinations

• Proximity ligation assay (PLA) adaptation:

  • Modify standard PLA protocols to incorporate the FITC-conjugated antibody

  • Use a complementary PLA probe that recognizes rabbit IgG

  • This allows visualization of protein-protein interactions within 40 nm distance

• FRET analysis considerations:

  • When pairing with cyan fluorescent protein (CFP)-tagged constructs, FITC is suitable as a FRET acceptor

  • For FRET with yellow fluorescent protein (YFP)-tagged constructs, FITC can serve as a donor

• Live-cell co-localization optimization:

  • For membrane-permeable applications, reduce antibody concentration (typically 1:500) and extend incubation times

  • Include appropriate controls for non-specific uptake

• Quantitative co-localization analysis:

  • Calculate Pearson's or Manders' coefficients between CSNK1G3 and potential interaction partners

  • Apply appropriate thresholding methods based on control samples

What are the critical parameters for optimizing FITC-conjugated CSNK1G3 antibody use in flow cytometry?

When optimizing FITC-conjugated CSNK1G3 antibody for flow cytometry applications, consider these critical parameters:

• Cell preparation optimization:

  • For intracellular detection, compare fixation methods (0.5-4% paraformaldehyde) and permeabilization agents (0.1% saponin, 0.1% Triton X-100, 90% methanol)

  • Standardize cell concentration to 1×10⁶ cells/mL for consistent results

  • Include viability dye (non-overlapping with FITC) to exclude dead cells that may bind antibodies non-specifically

• Titration and signal-to-noise optimization:

  • Create an antibody titration series (typically 0.1-10 μg/mL) plotted against signal-to-noise ratio

  • Determine optimal concentration at the inflection point before saturation

  • Compare staining index across conditions

• Compensation requirements:

  • Prepare single-color FITC control using the CSNK1G3 antibody or alternative FITC-conjugated antibody with similar brightness

  • Include unstained and isotype controls

  • For multi-color panels, perform full compensation matrix

• Protocol adaptations for phospho-epitopes:

  • If phosphorylation state is important, include phosphatase inhibitors (2 mM sodium orthovanadate, 10 mM sodium fluoride)

  • Compare basal vs. stimulated conditions

  • Consider methanol permeabilization for improved phospho-epitope detection

• Acquisition parameters:

  • Set PMT voltage to position negative population in first decade of logarithmic scale

  • Adjust FSC/SSC to properly identify cell populations

  • Collect sufficient events (minimum 10,000, ideally 50,000+) for statistical validity

• Analysis considerations:

  • Gate on singlets, viable cells, and relevant populations

  • Compare median fluorescence intensity rather than mean when population distributions are not normal

  • Consider biaxial plots of CSNK1G3 vs. known markers or interaction partners

How can I determine if the FITC conjugation affects the CSNK1G3 antibody's epitope recognition or binding kinetics?

Evaluating the impact of FITC conjugation on antibody performance is essential for accurate data interpretation:

• Parallel comparison methodology:

  • Design experiments using both conjugated and unconjugated versions of the same CSNK1G3 antibody clone

  • Implement identical protein concentrations, incubation times, and detection systems (using FITC-conjugated secondary antibody for the unconjugated version)

  • Compare signal intensities, background levels, and specificity patterns

• Epitope accessibility assessment:

  • Test antibody performance using varied fixation and permeabilization protocols

  • More pronounced differences between conjugated and unconjugated versions under certain conditions may indicate epitope masking

  • Analyze subcellular localization patterns for discrepancies

• Binding kinetics quantification:

  • Measure association and dissociation rates using surface plasmon resonance

  • Compare kinetics of conjugated vs. unconjugated antibodies against recombinant CSNK1G3

  • Determine if conjugation alters affinity (KD) values

• Competitive binding analysis:

  • Pre-incubate samples with unlabeled antibody before adding FITC-conjugated version

  • Perform with varying ratios to establish displacement curves

  • Compare EC50 values between competitive assays using conjugated vs. unconjugated competitors

• Cross-linking effects evaluation:

  • Assess if multiple FITC molecules per antibody (typical F/P ratios range from 3:1 to 7:1) affect cross-linking behaviors

  • Compare data using antibodies with different F/P ratios if available

  • Monitor for artifactual aggregation in membrane proteins

• pH sensitivity comparison:

  • Test antibody performance across pH range (5.5-8.0)

  • FITC fluorescence is pH-sensitive, potentially complicating interpretation in acidic compartments

What are common causes of weak or no signal when using FITC-conjugated CSNK1G3 antibody, and how can these issues be resolved?

Several factors can contribute to weak or absent signal when using FITC-conjugated CSNK1G3 antibody:

• Fluorophore degradation resolution:

  • FITC is sensitive to photobleaching; minimize light exposure during storage and experimental procedures

  • Check fluorophore integrity using spectrophotometry (FITC peak absorption ~495 nm)

  • If degraded, use a fresh aliquot and consider switching to more photostable alternatives (Alexa Fluor 488)

• Fixation-induced epitope masking solutions:

  • Test alternative fixation methods (cross-linking vs. precipitating fixatives)

  • Implement epitope retrieval techniques (heat-induced at 95°C in citrate buffer pH 6.0 or enzymatic with proteinase K)

  • Reduce fixation time to minimize over-fixation

• Expression level considerations:

  • CSNK1G3 may be expressed at low levels in certain cell types; increase exposure time within reasonable limits

  • Amplify signal using biotin-streptavidin systems or tyramide signal amplification

  • Consider cell types with known higher expression (consult gene expression databases)

• Antibody concentration optimization:

  • Initial testing should include a broad range (1:50 to 1:1000 dilutions)

  • For low-abundance targets, use higher concentrations (1:50 to 1:100)

  • Extend incubation time (overnight at 4°C) to improve signal

• Buffer and pH adjustments:

  • FITC fluorescence is optimal at slightly alkaline pH; ensure buffers are at pH 7.4-8.0

  • Add 10 mM HEPES buffer to maintain pH stability during long incubations

  • Test different permeabilization reagents that may better preserve epitope structure

• Detergent effects mitigation:

  • High detergent concentrations may extract membrane proteins; reduce Triton X-100 to 0.01% or switch to digitonin (0.005-0.01%)

  • For cytoskeletal proteins, use extraction buffers that preserve structural integrity

How should I address high background issues when using FITC-conjugated CSNK1G3 antibody in tissue sections or cell cultures?

High background is a common challenge with immunofluorescence; implement these strategies to improve signal-to-noise ratio:

• Tissue-specific autofluorescence reduction:

  • For tissues with high autofluorescence (brain, kidney, liver):

    • Treat with 0.1-1% sodium borohydride in PBS for 10 minutes before blocking

    • Apply 0.1-0.3% Sudan Black B in 70% ethanol after antibody incubation

    • Consider spectral unmixing during image acquisition

• Blocking optimization:

  • Test different blocking agents (5% normal serum, 3-5% BSA, commercial blockers)

  • Include 0.1-0.3% Triton X-100 in blocking buffer to reduce hydrophobic interactions

  • Add 0.1-0.3% glycine to quench unreacted aldehyde groups after fixation

  • Extend blocking time to 2 hours at room temperature or overnight at 4°C

• Non-specific binding reduction:

  • Include 5% serum from the host species of any secondary antibodies used

  • Add 0.1-0.5 M NaCl to reduce ionic interactions

  • Pre-adsorb antibody with acetone powder from tissues of unrelated species

• Wash protocol optimization:

  • Increase wash durations (5×10 minutes instead of standard 3×5 minutes)

  • Add 0.05% Tween-20 to wash buffers to remove weakly bound antibodies

  • Perform all washes with gentle agitation

• Antibody dilution adjustment:

  • Paradoxically, too concentrated antibody often increases background; test more dilute solutions

  • Optimal dilution is often more dilute than manufacturer's recommendation for indirect detection methods

• Fluorophore considerations:

  • FITC can have higher background in certain tissues due to autofluorescence in the green spectrum

  • Consider switching to red or far-red fluorophores for tissues with high green autofluorescence

What strategies can improve detection of low-abundance CSNK1G3 in specific cell types or subcellular compartments?

Detecting low-abundance CSNK1G3 requires specialized approaches:

• Signal amplification technologies:

  • Implement tyramide signal amplification (TSA), which can increase sensitivity 10-100 fold

  • Consider rolling circle amplification for extreme sensitivity requirements

  • Use avidin-biotin amplification systems with biotinylated primary or secondary antibodies

• Advanced microscopy techniques:

  • Apply deconvolution algorithms to improve signal-to-noise ratio

  • Utilize structured illumination microscopy (SIM) for improved resolution

  • Consider stimulated emission depletion (STED) microscopy for superior resolution of subcellular structures

• Sample preparation enhancements:

  • Optimize fixation specifically for CSNK1G3 preservation (compare cross-linkers vs. precipitating fixatives)

  • Implement antigen retrieval methods optimized for phospho-epitopes if applicable

  • Consider specialized permeabilization protocols for nuclear proteins

• Enrichment before detection:

  • Perform subcellular fractionation to enrich compartments of interest

  • Use proximity ligation assay (PLA) to detect CSNK1G3 interacting with known binding partners

  • Consider click chemistry approaches for nascent protein labeling

• Image acquisition optimization:

  • Increase exposure time within reasonable limits to avoid photobleaching

  • Implement frame averaging (4-8 frames) to improve signal-to-noise ratio

  • Use confocal microscopy with optimized pinhole settings (0.8-1.0 Airy units)

  • Consider EM-CCD cameras for improved sensitivity in low-light conditions

• Analysis enhancements:

  • Apply background subtraction algorithms appropriate for the specific sample

  • Implement deconvolution for improved signal quality

  • Use advanced segmentation algorithms for accurate quantification of subcellular structures

How does CSNK1G3 function compare to other CSNK1G family members (CSNK1G1 and CSNK1G2) in experimental systems?

Understanding the functional differences between casein kinase 1 gamma family members is crucial for accurate data interpretation:

• Distinctive roles in signaling pathways:

  • Unlike CSNK1G2, CSNK1G3 does not strongly suppress RIPK3 kinase activity and necroptosis

  • CSNK1G2 binds to RIPK3 through auto-phosphorylation at serine 211 and threonine 215 sites, a mechanism not observed with CSNK1G3

  • While sharing sequence homology, each family member appears to have distinct protein interaction partners and substrates

• Expression pattern differences:

  • CSNK1G3 shows distinct tissue distribution patterns compared to CSNK1G1 and CSNK1G2

  • When studying cell type-specific functions, consider relative expression levels of all family members

  • Create experimental controls that assess activity of all three family members in parallel

• Substrate specificity considerations:

  • Despite high homology in catalytic domains, substrate preferences differ

  • When investigating phosphorylation events, validate specific contributions of each family member

  • Consider using specific inhibitors or CRISPR knockout models to distinguish functions

• Localization differences:

  • Compare subcellular localization patterns using specific antibodies against each family member

  • Assess co-localization with organelle markers to identify compartment-specific roles

  • Membrane association patterns may differ between family members due to variations in regulatory domains

• Response to cellular stress:

  • CSNK1G2 is implicated in stress responses including necroptosis regulation, while CSNK1G3's roles may differ

  • Compare phosphorylation status and localization under various stress conditions

  • Assess potential compensatory mechanisms when one family member is depleted

What controls are essential when using FITC-conjugated CSNK1G3 antibody to ensure reliable quantitative analysis?

Implementing appropriate controls is critical for quantitative immunofluorescence or flow cytometry:

• Essential negative controls:

  • Isotype control: FITC-conjugated rabbit IgG at matching concentration

  • Secondary antibody only (if indirect method used in parallel)

  • Blocking peptide competition: pre-incubation of antibody with immunizing peptide

  • Genetic controls: CRISPR knockout or siRNA knockdown samples

• Positive control implementation:

  • Cell lines or tissues with verified CSNK1G3 expression

  • Overexpression systems with tagged CSNK1G3

  • Treatment conditions known to upregulate CSNK1G3 expression or activation

• Technical controls for quantification:

  • Fluorescence calibration beads for standardizing intensity measurements

  • Internal reference standards for normalization across experiments

  • Standard curves using recombinant protein when applicable

• Autofluorescence assessment:

  • Unstained samples to establish baseline autofluorescence

  • Single-color controls for spectral overlap compensation

  • Matched filter sets appropriate for FITC (excitation ~495 nm, emission ~520 nm)

• Methodology validation controls:

  • Process controls (samples processed identically except for primary antibody)

  • Technical replicates to assess methodological variability

  • Biological replicates to assess biological variability

  • Alternate detection methods (e.g., Western blot) for validation

• Image acquisition controls:

  • Identical exposure settings across comparable samples

  • Point spread function measurements using sub-resolution beads

  • Flatfield correction for uneven illumination

• Analysis validation:

  • Blinded analysis by multiple observers

  • Multiple quantification algorithms compared

  • Explicit threshold determination methods