CALR Antibody, Biotin conjugated

What is CALR (Calreticulin) and what cellular functions does it perform?

Calreticulin (CALR) is a multifunctional protein initially characterized as an endoplasmic reticulum resident protein. It functions primarily as a calcium-binding chaperone that promotes protein folding, oligomeric assembly, and quality control in the endoplasmic reticulum through the calreticulin/calnexin cycle. CALR interacts transiently with monoglucosylated glycoproteins synthesized in the ER. Beyond this classical role, CALR has been identified in multiple cellular locations including the cytoplasm, cell membrane, and extracellular matrix. It also functions in nuclear export regulation, particularly by interacting with the DNA-binding domain of NR3C1 and mediating its nuclear export. In oocyte development, CALR participates in maternal gene expression regulation and may regulate calcium homeostasis during oocyte maturation .

Research has revealed CALR's expanding biological significance beyond its chaperone function. Initially considered solely a resident protein of the sarcoplasmic reticulum with Ca²⁺ buffer and molecular chaperone characteristics, CALR has gained attention for potential roles in tumor progression and anti-tumor immunity. Studies have shown that conditioned medium containing CALR released from cultured cells can kill tumor cells and reduce angiogenesis. Additional research suggests CALR may participate in tumor cell clearance by activating the immune system .

What are the common applications for CALR Antibody, Biotin conjugated?

CALR Antibody, Biotin conjugated is utilized across several research applications, with specific protocols optimized for each methodology:

Biotin conjugation offers advantages in these applications by enabling versatile detection systems through strong avidin/streptavidin binding. This conjugation allows for signal amplification in detection systems, making it particularly valuable when studying proteins expressed at lower levels. When using biotin-conjugated antibodies in multi-step detection protocols, researchers should optimize blocking steps to minimize background staining that can result from endogenous biotin in certain tissues .

What are the optimal storage conditions and shelf life for CALR Antibody, Biotin conjugated?

For maximum stability and performance of CALR Antibody, Biotin conjugated, the following storage conditions are recommended:

• Store at -20°C for long-term storage (up to 12 months from manufacture)

• Avoid repeated freeze-thaw cycles which can damage antibody activity

• Upon receipt of a liquid formulation, aliquot into smaller volumes if repeated use is anticipated

Most commercial CALR Antibody, Biotin conjugated products are supplied in a storage buffer containing:

• 0.01M TBS (pH 7.4) or PBS (pH 7.4)

• 1% BSA (acts as a stabilizer)

• 0.03% Proclin300 (preservative)

• 50% Glycerol (prevents freezing damage)

For reconstituted lyophilized standards or antibodies, manufacturers generally do not recommend reuse after the initial reconstitution . Opened products should ideally be used within one month, even when properly stored .

How should researchers validate the specificity of CALR Antibody, Biotin conjugated?

Validating antibody specificity is a critical methodological step for ensuring experimental reliability. For CALR Antibody, Biotin conjugated, researchers should implement a multi-faceted validation approach:

• Western blot analysis: Confirm a single band of appropriate molecular weight (approximately 55-60 kDa for CALR). The search results include validation images showing successful detection of CALR in human placenta tissue lysate .

• Positive and negative controls:

  • Use tissues or cell lines known to express CALR (positive control)

  • Compare with CALR-knockout or low-expressing samples (negative control)

  • Include isotype controls to identify non-specific binding

• Cross-reactivity assessment: Review manufacturer specifications regarding species reactivity and predicted reactivity. Available CALR antibodies show reactivity with human, mouse, and rat CALR, with predicted reactivity against dog, cow, pig, horse, chicken, rabbit, and guinea pig CALR depending on the specific product .

• Blocking peptide competition: When available, use the immunogen peptide to pre-absorb the antibody before application, which should eliminate specific binding.

• Multiple antibody verification: Compare results using antibodies raised against different epitopes of CALR to confirm specificity.

When publishing results, researchers should report complete validation methods and include antibody catalog numbers, lot numbers, and dilutions to ensure reproducibility.

How can CALR Antibody, Biotin conjugated be used effectively in cancer research?

CALR has emerged as a significant molecule in cancer research, with bioinformatics analyses suggesting its potential as both a biomarker for prognosis prediction and a target for tumor molecular and immunotherapy . When using CALR Antibody, Biotin conjugated in cancer research, researchers should consider:

• Expression analysis across cancer types:

  • CALR expression varies across different tumor types, making it important to establish baseline expression in both normal and malignant tissues for your specific cancer model

  • Bioinformatics analyses have been conducted to assess CALR expression in pan-cancer studies

• Correlation with clinical outcomes:

  • Survival analysis linking CALR expression with clinical prognosis should be performed

  • Research has shown correlations between CALR expression levels and clinical prognosis in multiple cancer types

• Immune infiltration studies:

  • CALR plays roles in tumor immune regulation across multiple mechanisms

  • Studies using TIMER database analysis have revealed correlations between CALR and six kinds of immune cell infiltrations (B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and DCs) in 39 types of tumors

  • Particularly strong correlations were found in breast cancer and colon cancer, suggesting CALR's carcinogenic mechanism may be related to anti-tumor immunity

• Methodological approaches:

  • Immunohistochemistry with biotin-conjugated CALR antibodies can visualize expression patterns in tumor tissues

  • Flow cytometry can quantify CALR expression on tumor cell surfaces

  • Western blot analysis can assess total protein levels across different cancer cell lines

When designing cancer research experiments using CALR Antibody, Biotin conjugated, researchers should carefully select appropriate controls and consider the subcellular localization of CALR (cytoplasm, secreted, cell membrane, extracellular matrix) in their specific tumor model .

What methodological considerations should be addressed when using CALR Antibody, Biotin conjugated in immunological studies?

CALR plays significant roles in immune regulation, particularly in tumor immunology. When designing immunological studies using CALR Antibody, Biotin conjugated, researchers should consider these methodological approaches:

• Immune cell infiltration analysis:

  • CALR has been shown to correlate with multiple immune cell types in tumor microenvironments

  • Research indicates correlation between CALR and 4 kinds of immune cells in breast cancer and colon cancer (p<0.05)

  • Consider co-staining for CALR alongside immune cell markers to assess potential interactions

• Protocol optimization for immune tissues:

  • When studying lymphoid tissues, researchers should implement additional blocking steps to reduce non-specific binding

  • Endogenous biotin in some immune cells may produce false positive signals; biotin blocking kits should be employed

  • Fixation methods should be optimized as overfixation can mask CALR epitopes

• CALR and immune regulation mechanisms:

  • CALR can participate in tumor cell clearance by activating the immune system

  • When analyzing these pathways, consider designing experiments that assess both membrane-associated and secreted CALR

  • The biotin conjugation allows for sensitive detection in complex immune microenvironments

• Multiplexed imaging approaches:

  • Biotin-conjugated antibodies can be particularly useful in multiplexed immunostaining protocols

  • When designing such experiments, carefully select complementary fluorophores attached to streptavidin to avoid spectral overlap

  • Controls for confirming specificity in multiplexed formats are essential

When documenting results, researchers should report complete methodological details including blocking reagents, incubation times, wash procedures, and detection systems to enable reproducibility .

How do biotin-conjugated antibodies perform differently than other conjugates in CALR detection systems?

Biotin-conjugated CALR antibodies offer distinct advantages and limitations compared to other conjugate types:

The biotin-streptavidin system provides significant signal amplification because each biotin-conjugated antibody can bind multiple streptavidin molecules, each carrying multiple reporter molecules (HRP enzymes or fluorophores) . This makes biotin conjugation particularly valuable for detecting CALR in tissues or applications where signal enhancement is needed.

For optimal performance, biotinylation kits used by manufacturers are specifically designed to create high-quality conjugates while minimizing assay background . When designing experiments, researchers may need to test different dilutions of the working biotin conjugate antibody to optimize signal-to-noise ratio .

What are common troubleshooting approaches for weak or no signal when using CALR Antibody, Biotin conjugated?

When researchers encounter weak or absent signals when using CALR Antibody, Biotin conjugated, a systematic troubleshooting approach should be implemented:

• Antibody concentration:

  • Verify optimal dilution ranges for your specific application

  • For Western blot, try concentrations between 1:300-5000 or 1:500-2000

  • For ELISA, try 1:500-1000

  • For IHC-P, try 1:200-400

  • For IHC-F, try 1:100-500

• Detection system optimization:

  • Ensure streptavidin-HRP or streptavidin-fluorophore is functional

  • Consider increasing concentration of detection reagent

  • For Western blots, extend exposure time or use more sensitive substrate

  • For ELISA, verify proper preparation of working Biotin Conjugate Antibody by diluting 1:100 of Concentrated Biotin Conjugate Antibody with appropriate diluent

• Sample preparation issues:

  • CALR is normally located in the cytoplasm, secreted fraction, cell membrane, and extracellular matrix

  • Ensure extraction method is appropriate for isolating CALR

  • Check protein quantification to ensure adequate loading

  • Verify pH and ionic strength of buffers, as these can affect antibody-antigen binding

• Antigen retrieval for IHC:

  • If using paraffin sections, optimize antigen retrieval methods (heat vs. enzymatic)

  • Extend retrieval time if signal is weak

  • CALR epitopes may be sensitive to particular fixation methods

• Blocking optimization:

  • For tissues with high endogenous biotin (liver, kidney, brain), use specialized biotin blocking kits

  • Insufficient blocking can lead to high background that obscures specific signal

  • Try alternative blocking reagents if standard protocols fail

If signal remains weak after these optimizations, consider testing the antibody on positive control samples with known high CALR expression, such as human placenta tissue lysate , to determine if the issue is with the antibody or the experimental samples.

What are the best approaches for using CALR Antibody, Biotin conjugated in multiplex detection systems?

Multiplex detection systems allow researchers to simultaneously visualize multiple targets, increasing the data obtained from valuable samples. When incorporating CALR Antibody, Biotin conjugated into multiplex protocols:

• Strategic antibody selection:

  • Choose primary antibodies raised in different host species to prevent cross-reactivity

  • When using rabbit-derived CALR Antibody, Biotin conjugated , pair with mouse, goat, or rat antibodies for other targets

  • Verify that secondary detection systems don't cross-react with non-target primaries

• Order of application optimization:

  • When CALR is abundant, apply the CALR Antibody, Biotin conjugated later in the staining sequence

  • For weaker CALR expression, apply earlier with longer incubation times

  • Test different staining sequences to minimize interference between detection systems

• Spectral considerations for fluorescent detection:

  • Select streptavidin conjugated to fluorophores with minimal spectral overlap with other fluorophores in the multiplex panel

  • Include proper single-stain controls to facilitate spectral unmixing if needed

  • Consider photobleaching characteristics when designing imaging protocols

• Sequential detection techniques:

  • For challenging multiplex combinations, consider sequential rounds of staining with stripping/blocking between rounds

  • Document complete removal of previous detection reagents before applying new ones

  • Specialized multiplex protocols may be required for formalin-fixed tissues

• Validation and controls:

  • Always include single-stain controls alongside multiplex samples

  • Compare multiplex staining patterns with those obtained in single-stain experiments

  • Consider computational approaches to resolve potential spatial overlap

For quantitative analyses of multiplex data, establish clear thresholds for positive staining and use digital image analysis to ensure consistent quantification across samples and experimental batches.

How should researchers approach epitope blocking and endogenous biotin interference when using CALR Antibody, Biotin conjugated?

Endogenous biotin and non-specific binding can significantly impact the reliability of experiments using CALR Antibody, Biotin conjugated. Researchers should implement tailored blocking strategies:

• Endogenous biotin blocking:

  • Tissues like liver, kidney, brain, and adipose tissue contain high levels of endogenous biotin

  • Commercial avidin/biotin blocking kits should be used prior to antibody application

  • Sequential application of avidin (to block endogenous biotin) followed by biotin (to block remaining avidin sites)

  • Extended blocking times may be required for tissues with particularly high biotin content

• Non-specific binding prevention:

  • For CALR detection, blocking buffers containing 1% BSA are typically effective

  • Ensure blocking buffer pH matches recommended antibody incubation conditions (typically pH 7.4)

  • Consider including 0.1-0.3% Triton X-100 in blocking solutions for intracellular applications

  • For tissues with high background, try alternative blocking proteins (casein, non-fat dry milk)

• Cross-reactivity mitigation:

  • Review antibody specificity data and avoid tissues with known cross-reactivity

  • Include isotype controls at the same concentration as the primary antibody

  • Consider absorption controls using the immunizing peptide when available

  • For multiplexed applications, test each antibody individually before combining

• Protocol optimization:

  • Increase wash steps (number and duration) after antibody incubation

  • Dilute antibody in blocking buffer rather than plain buffer solution

  • Optimize incubation temperature (4°C overnight may yield better specificity than room temperature incubation)

  • Consider reduced permeabilization for membrane CALR detection to limit access to intracellular biotin

By systematically addressing these factors, researchers can significantly improve signal-to-noise ratio and ensure that observed staining truly represents CALR distribution rather than technical artifacts.

How is CALR Antibody, Biotin conjugated being used to investigate CALR's role in cancer immunotherapy?

Recent research has highlighted CALR's potential significance in cancer immunotherapy, making CALR Antibody, Biotin conjugated an important tool for investigating these mechanisms:

• CALR as an immunotherapy target:

  • Bioinformatics analyses suggest CALR may serve as a potential target for tumor immunotherapy

  • CALR Antibody, Biotin conjugated enables detection of CALR on tumor cell surfaces, which may correlate with immunotherapy response

  • Researchers can use this antibody to assess whether modulating CALR exposure affects immune recognition of tumor cells

• Immune cell infiltration correlation:

  • Studies using TIMER database analysis have revealed correlations between CALR expression and six types of immune cell infiltrations across 39 tumor types

  • CALR Antibody, Biotin conjugated can be used in co-staining experiments to visualize CALR in relation to tumor-infiltrating immune cells

  • Particularly strong correlations exist in breast cancer and colon cancer, suggesting these might be priority tumor types for CALR-targeted immunotherapy investigations

• Methodological approaches:

  • Flow cytometry with CALR Antibody, Biotin conjugated can quantify surface CALR exposure on live tumor cells

  • Immunohistochemistry can visualize spatial relationships between CALR-expressing cells and immune infiltrates

  • Proximity ligation assays using biotinylated antibodies can detect CALR interactions with immune receptors

  • Sequential multiplex immunofluorescence allows visualization of complex cellular relationships in the tumor microenvironment

• Research findings supporting immunotherapy connections:

  • Studies have found that CALR may participate in tumor cell clearance by activating the immune system

  • Conditioned medium containing CALR released from cultured cells can kill tumor cells and reduce angiogenesis

  • These findings suggest CALR's exposure or release may be manipulated to enhance anti-tumor immunity

Researchers designing immunotherapy-related experiments should consider both membrane-associated and secreted forms of CALR, as both may play roles in immune system interactions .

What technical considerations should be addressed when using CALR Antibody, Biotin conjugated in single-cell analysis techniques?

As single-cell analysis techniques become increasingly important in understanding heterogeneous cellular responses, researchers need specific protocols for using CALR Antibody, Biotin conjugated in these applications:

• Flow cytometry optimization:

  • Titrate CALR Antibody, Biotin conjugated to determine optimal concentration for specific cell types

  • Use streptavidin conjugated to bright fluorophores (PE, APC) for optimal signal separation

  • Include FMO (Fluorescence Minus One) controls to establish proper gating strategies

  • For intracellular CALR, ensure permeabilization protocols maintain epitope integrity

• Mass cytometry (CyTOF) considerations:

  • When adapting protocols for mass cytometry, use metal-conjugated streptavidin

  • Optimize staining concentration to avoid signal spillover into adjacent mass channels

  • Consider signal amplification strategies for low-abundance CALR detection

  • Compare surface and intracellular staining patterns to distinguish localization

• Single-cell imaging applications:

  • For imaging flow cytometry, adjust exposure settings to capture CALR localization

  • In highly multiplex imaging, place CALR in a detection channel with minimal spillover

  • Consider photobleaching characteristics if performing time-lapse imaging

  • For super-resolution microscopy, use small streptavidin-fluorophore conjugates to minimize linkage error

• Single-cell sequencing integration:

  • For CITE-seq or similar approaches, ensure biotinylated antibodies are compatible with oligonucleotide-tagged streptavidin

  • Validate that antibody binding doesn't affect RNA quality for downstream analysis

  • Consider cell fixation impacts on both protein detection and RNA recovery

  • Include spike-in controls for antibody quantification normalization

• Protocol modifications for rare cell populations:

  • For detecting CALR in rare cells, consider magnetic enrichment with streptavidin beads before analysis

  • Implement doublet discrimination strategies to ensure single-cell resolution

  • Use viability dyes to exclude dead cells that may bind antibodies non-specifically

  • Consider pre-enrichment of target populations when CALR+ cells are rare

These technical considerations ensure that CALR Antibody, Biotin conjugated performs optimally in single-cell analysis workflows, enabling reliable detection at both population and individual cell levels .