CACNA1C Antibody, FITC conjugated

What is CACNA1C Antibody, FITC conjugated and what does it target?

CACNA1C Antibody, FITC conjugated is a polyclonal antibody raised in rabbits that specifically targets the Voltage-dependent L-type calcium channel subunit alpha-1C protein. This protein is also known by several aliases including Calcium channel, L type, alpha-1 polypeptide, isoform 1, cardiac muscle, and Voltage-gated calcium channel subunit alpha Cav1.2 . The antibody recognizes the human CACNA1C protein, specifically the region corresponding to amino acids 1755-1971 of the recombinant Human Voltage-dependent L-type calcium channel subunit alpha-1C protein . The FITC conjugation allows for direct fluorescent detection in various applications without requiring secondary antibodies.

What are the optimal storage conditions for CACNA1C Antibody?

For maximum stability and activity retention, CACNA1C Antibody, FITC conjugated should be stored at either -20°C or -80°C immediately upon receipt . The antibody is supplied in a liquid form containing preservative (0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4) . It is critical to avoid repeated freeze-thaw cycles as this can lead to protein denaturation and loss of antibody activity . For working solutions, aliquoting the antibody into single-use volumes before freezing is recommended to minimize freeze-thaw cycles.

What applications is CACNA1C Antibody validated for?

According to the product information, CACNA1C Antibody, FITC conjugated has been validated for ELISA applications . Additional antibody variants of CACNA1C (non-FITC conjugated) have been validated for immunohistochemistry (IHC) with a recommended dilution ratio of 1:25-1:100 . In research studies, CACNA1C antibodies have been successfully utilized in multiple applications including:

• Flow cytometry (at 1μg per 10^6 cells)

• Immunofluorescence (at 1:50 dilution)

• Western blotting (at 1:500 dilution)

• Co-immunoprecipitation for protein interaction studies

What is the purification method and quality control for this antibody?

The CACNA1C Antibody, FITC conjugated is purified using Protein G affinity chromatography with a purification level greater than 95% . The antibody is of IgG isotype and is derived from a polyclonal preparation, meaning it recognizes multiple epitopes on the target protein . Quality control specifications typically include verification of specificity through Western blotting or ELISA against the immunogen peptide, as well as confirmation of the fluorophore conjugation efficiency.

How does CACNA1C expression relate to rituximab resistance in lymphoma treatment?

Research has identified a significant inverse correlation between CACNA1C expression and resistance to R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) therapy in diffuse large B-cell lymphoma (DLBCL) . Multiple independent cohort studies have shown that CACNA1C expression serves as an independent prognostic factor for R-CHOP resistance, even after adjusting for other factors such as International Prognostic Index, cell of origin classification, and MYC/BCL2 double expression .

Mechanistically, CACNA1C has been demonstrated to directly interact with CD20 (the target of rituximab), contributing to CD20 stabilization on the cell surface . Loss of CACNA1C expression reduces rituximab-induced apoptosis and tumor shrinkage. Expression analysis revealed that CACNA1C mRNA levels were significantly lower in rituximab-resistant patients (p=0.009), while interestingly, CD20 mRNA expression did not show significant differences between sensitive and resistant groups .

These findings suggest that CACNA1C expression assessment could potentially serve as a predictive biomarker for rituximab therapy response, and that targeting L-type calcium channel expression or activity might provide a strategy to overcome rituximab resistance.

What methodologies are optimal for detecting CACNA1C protein in tissue and cell samples?

Several methodologies have been validated for detecting CACNA1C protein in research settings:

For tissue sections (Immunohistochemistry):

• Use 5μm thick paraffin sections

• Perform antigen retrieval by high-pressure heating for 1 minute in pH 6.0 buffer

• Apply CACNA1C antibody at 1:100 dilution

• Define positive staining as more than 30% of lymphoma cells showing membrane localization

For cell suspensions (Flow cytometry):

• Fix cells with 80% methanol for 5 minutes

• Permeabilize with PBST (0.2% Tween-20) for 20 minutes

• Block with 10% normal goat serum

• Incubate with CACNA1C antibody (1μg per 10^6 cells) for 30 minutes at 22°C

• Apply appropriate fluorochrome-conjugated secondary antibody (unless using directly conjugated antibody)

For protein detection (Western blotting):

• Extract total proteins with NP40 lysis buffer

• Use CACNA1C antibody at 1:500 dilution

• Visualize using enhanced chemiluminescence systems

For co-localization studies (Immunofluorescence):

• Fix and permeabilize cells appropriately

• Incubate with CACNA1C antibody (1:50 dilution) overnight at 4°C

• Use fluorophore-conjugated secondary antibodies (e.g., Cy3 at 1:1000)

• Counterstain nuclei with DAPI

• Mount with appropriate fluorescence mounting medium

• Image using confocal microscopy

How can the interaction between CACNA1C and CD20 be analyzed in experimental systems?

The interaction between CACNA1C and CD20 can be studied using several complementary approaches:

Co-immunoprecipitation (Co-IP):

• Extract total proteins from cells (e.g., OCI-ly7 cells) using NP40 lysis buffer

• Incubate cell lysate with anti-CD20 antibody (1:50 dilution) or control IgG overnight at 4°C on a rotating shaker

• Add protein A/G PLUS-Agarose (20μl per 500μl lysate) and incubate for 3 hours at 4°C

• Wash the pellets three times with NP40 lysis buffer

• Analyze pulled-down proteins by Western blotting using anti-CACNA1C antibody (1:500 dilution)

Co-localization by confocal microscopy:

• Culture cells with or without treatment (e.g., rituximab at 50μg/ml)

• Incubate with antibodies against CD20 (1:50) and CACNA1C (1:50) at 4°C overnight

• Apply appropriate fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 647 and Cy3 at 1:1000 dilution)

• Counterstain with DAPI (1μg/ml) for 1 minute

• Mount with fluorescence mounting medium

• Visualize and analyze co-localization using confocal microscopy

Functional studies:

• Manipulate CACNA1C expression using shRNA or overexpression constructs

• Assess effects on CD20 protein levels by Western blotting

• Evaluate impact on rituximab-induced calcium flux and apoptosis

• Compare findings in both in vitro cell culture and in vivo xenograft models

What is the relationship between calcium flux and CACNA1C function in rituximab response?

CACNA1C encodes an L-type calcium channel that mediates calcium influx upon membrane polarization. Research has shown that this calcium signaling plays a critical role in rituximab-induced apoptosis . To study this relationship:

Calcium flux assay methodology:

• Suspend cells (e.g., OCI-ly3, OCI-ly7) at 1×10^6 cells/ml in HBSS solution containing 1% BSA

• Load with 2μM Fluo-4 AM calcium indicator for 30 minutes at 37°C in the dark

• Wash twice with HBSS solution

• Resuspend in saline solution containing 10mM HEPES, 1mM Na₂HPO₄, 137mM NaCl, 5mM KCl, 1mM CaCl₂, 0.5mM MgCl₂, 5mM Glucose, 0.1% BSA

• Add rituximab (50μg/ml) and immediately measure calcium flux using flow cytometry

• Monitor Fluo-4 fluorescence (peak at 526 nm) in FL1 channel

Findings indicate that functional modulators of L-type calcium channels alter rituximab-induced apoptosis and tumor suppression, suggesting that CACNA1C-mediated calcium flux is a critical mechanism in rituximab response . This relationship provides potential opportunities for therapeutic intervention to enhance rituximab efficacy through calcium channel modulation.

How can CACNA1C expression be manipulated in experimental models?

For research requiring modulation of CACNA1C expression, several approaches have been validated:

RNA interference using shRNA:

• Design shRNA to target CACNA1C mRNA

• Clone into appropriate lentiviral vectors (e.g., pLKO.1-puro digested with AgeI/EcoRI)

• Generate lentivirus by co-transfecting 293T cells with:

  • 12μg of shRNA-containing plasmid

  • 8μg of packing vector pSPAX2

  • 4μg of envelope vector pMD2G

• Collect viral supernatant after 48 hours and concentrate using PEG 6000

• Transduce target cells overnight

• Select transduced cells using puromycin (2μg/ml)

MicroRNA-based regulation:
Research has identified that CACNA1C expression is directly regulated by miRNA-363, whose high expression is associated with worse prognosis in DLBCL . This relationship can be exploited by:

• Generating lentiviral vectors expressing miRNA-363 under the control of the Ubc promoter

• Transducing cells using the protocol described above

• Validating CACNA1C downregulation by Western blotting or qPCR

Functional studies:
The effects of CACNA1C modulation can be assessed in both in vitro and in vivo models:

• In vitro: Measure rituximab-induced apoptosis, calcium flux, and CD20 stability

• In vivo: Develop xenograft models in immunodeficient mice and measure tumor volume response to rituximab treatment

What controls should be included when using CACNA1C Antibody in experimental protocols?

When designing experiments utilizing CACNA1C Antibody, FITC conjugated, the following controls should be included:

For immunohistochemistry and immunofluorescence:

• Isotype control: Use rabbit IgG at the same concentration as the primary antibody

• Negative tissue control: Use tissues known to lack CACNA1C expression

• Positive tissue control: Include tissues with validated CACNA1C expression

• Omission control: Perform staining without primary antibody to assess background

For flow cytometry:

• Unstained cells to establish autofluorescence baseline

• Isotype control (rabbit IgG-FITC) to determine non-specific binding

• Single-color controls for compensation when performing multi-color analysis

• FMO (Fluorescence Minus One) controls for accurate gating

For Western blotting:

• Loading control (e.g., β-actin, GAPDH) to normalize protein amounts

• Positive control lysate from cells known to express CACNA1C

• Molecular weight marker to confirm target band size

For co-immunoprecipitation:

• Non-specific IgG control (anti-IgG) to identify non-specific binding

• Input sample (pre-IP lysate) to confirm presence of target proteins

• Reverse IP (using anti-CACNA1C to pull down CD20) to validate interaction

How can CACNA1C expression be accurately quantified in patient samples?

For clinical and translational research involving patient samples, several methods for quantifying CACNA1C expression have been validated:

mRNA expression analysis:

• Use specific probe sets (242973_at and 238636_at) for CACNA1C mRNA expression quantification

• Apply appropriate statistical methods to assess correlation with clinical outcomes

• Consider multivariate logistic regression to examine the effect of CACNA1C mRNA expression on treatment resistance while controlling for other variables

Protein expression in tissue samples:

• Perform immunohistochemistry using standardized protocols

• Define positive staining criteria (e.g., >30% of lymphoma cells showing membrane localization)

• Use digital image analysis for objective quantification

• Consider double-staining with CD20 to assess co-localization

Patient stratification:

What are common issues when using CACNA1C Antibody in immunofluorescence and how can they be resolved?

When using CACNA1C Antibody, FITC conjugated for immunofluorescence, researchers may encounter several challenges:

High background fluorescence:

• Increase blocking time with 10% normal goat serum

• Optimize antibody concentration (perform titration experiments)

• Ensure proper washing steps (3-5 washes of 5 minutes each)

• Use 0.2% Tween-20 in wash buffer to reduce non-specific binding

• Consider using Sudan Black B (0.1% in 70% ethanol) to reduce autofluorescence

Weak or absent signal:

• Verify antigen retrieval efficacy (use high-pressure heating in pH 6.0 buffer)

• Optimize antibody concentration (try 1:50 dilution as a starting point)

• Extend primary antibody incubation time (overnight at 4°C)

• Ensure proper sample fixation (80% methanol for 5 minutes)

• Verify that storage conditions haven't compromised antibody activity

Photobleaching:

• Minimize exposure to light during all steps

• Use anti-fade mounting medium

• Capture images promptly after staining

• Consider using stronger initial signal by optimizing antibody concentration

How can calcium flux assays be optimized when studying CACNA1C function?

Calcium flux assays are critical for studying CACNA1C function in relation to rituximab response. Optimization strategies include:

Cell preparation:

• Ensure consistent cell density (1×10^6 cells/ml) for reproducible results

• Use healthy cells in log-phase growth with >95% viability

• Avoid serum in assay buffer as it can interfere with calcium indicator loading

Calcium indicator loading:

• Optimize Fluo-4 AM concentration (starting with 2μM is recommended)

• Include 0.02% Pluronic F-127 to improve indicator solubility and loading

• Maintain consistent loading time (30 minutes at 37°C in the dark)

• Perform thorough washing to remove extracellular dye

Signal optimization:

• Establish baseline fluorescence before adding stimuli

• Add rituximab (50μg/ml) and immediately begin acquisition

• Maintain consistent temperature during acquisition (room temperature)

• Collect data at appropriate intervals (every 10 seconds for 5-10 minutes)

Analysis considerations:

• Calculate the ratio of stimulated to baseline fluorescence

• Compare peak heights and areas under curve

• Evaluate kinetics of calcium flux (time to peak, duration)

• Include appropriate controls (calcium ionophore as positive control)

What emerging applications of CACNA1C research might impact cancer therapy?

Research on CACNA1C has revealed several promising directions that may impact cancer therapy:

Predictive biomarker development:

• The inverse correlation between CACNA1C expression and rituximab resistance suggests potential use as a predictive biomarker for DLBCL patients receiving R-CHOP therapy

• Further validation in prospective clinical trials would be needed to establish clinical utility

Combination therapy approaches:

• L-type calcium channel modulators could potentially enhance rituximab efficacy

• Developing specific CACNA1C activators might overcome rituximab resistance

• Combination strategies targeting both CD20 and calcium signaling pathways may improve treatment outcomes

Understanding regulatory mechanisms:

• The discovery that miRNA-363 regulates CACNA1C expression opens possibilities for miRNA-based therapeutic approaches

• Developing strategies to counter miRNA-363 might restore CACNA1C expression and rituximab sensitivity

Expanding to other cancer types:

• Investigating CACNA1C's role in other B-cell malignancies and solid tumors

• Exploring calcium channel involvement in resistance to other targeted therapies beyond rituximab