TRIM3 Antibody, Biotin conjugated

What is TRIM3 and what cellular functions does it perform?

TRIM3, also known as Brain-expressed RING finger protein (BERP), RING finger protein 22 (RNF22), or RING finger protein 97 (RNF97), is a 744 amino acid protein belonging to the TRIM family characterized by the RING-B-box coiled-coil (RBCC) structure . TRIM3 functions as an E3 ubiquitin ligase playing essential roles in multiple cellular processes.

TRIM3's primary functions include:

• Vesicular trafficking via association with the CART complex, which is necessary for efficient transferrin receptor recycling

• Regulation of neuronal plasticity, learning, and memory through polyubiquitination of synaptic components

• Cytoskeletal regulation through interactions with α-actinin-4 and myosin V

• Cell motility regulation by positively regulating the microtubule-dependent motor protein KIF21B

• Cell cycle regulation via RING-dependent E3 ligase activity that can induce growth arrest

• Innate immune response modulation through TLR3-mediated signaling pathways

The ubiquitin ligase activity of TRIM3 ensures that misfolded or damaged proteins are correctly identified and directed towards degradation, which is essential for normal cell function .

What is biotin conjugation and why is it useful for antibody applications?

Biotin conjugation is a process where biotin molecules (vitamin B7, 240 Da) are covalently attached to antibodies, typically via primary amine groups (lysine residues). Usually, between 3 and 6 biotin molecules are conjugated to each antibody molecule .

This conjugation offers several significant advantages:

• Exceptional binding affinity: The biotin-(strept)avidin interaction has a dissociation constant (Kd) of 10^-14 to 10^-15, making it one of the strongest non-covalent interactions in nature - 1,000 to 1,000,000 times stronger than typical antigen-antibody interactions

• Signal amplification: The tetravalent structure of avidin/streptavidin allows multiple biotin-labeled detection molecules to bind, greatly enhancing signal strength

• Versatility: Biotin's relatively small size, flexible valeric side chain, and ease of conjugation make it well-suited for protein labeling without significantly altering antibody-antigen interaction

• Stability: The biotin-streptavidin complex demonstrates astonishing stability against proteolytic enzymes, temperature and pH extremes, and harsh organic reagents

• Compatibility: The system works efficiently with various detection methods including colorimetric, fluorescent, and chemiluminescent approaches

What applications are suitable for TRIM3 biotin-conjugated antibodies?

Based on manufacturer specifications, TRIM3 biotin-conjugated antibodies are suitable for various research applications:

For optimal research outcomes, verify the specific applications validated by the manufacturer for your particular TRIM3 biotin-conjugated antibody. Different antibody clones may perform differently across these applications based on epitope accessibility and antibody characteristics .

How should TRIM3 biotin-conjugated antibodies be stored to maintain optimal activity?

Proper storage is critical for maintaining the activity of TRIM3 biotin-conjugated antibodies. Based on manufacturer recommendations:

• Temperature: Store at -20°C or -80°C for long-term preservation

• Light protection: All conjugated antibodies should be stored in light-protected vials or covered with light-protecting material (e.g., aluminum foil) to prevent photobleaching of the biotin complex

• Freeze-thaw cycles: Avoid repeated freezing and thawing as this will compromise both enzyme activity and antibody binding capacity

• Buffer composition: Many commercial preparations are supplied in a protective buffer containing:

  • 50% Glycerol (cryoprotectant)

  • 0.01M PBS, pH 7.4 (physiological buffer)

  • 0.03% Proclin 300 (preservative)

• Extended storage recommendations: For storage beyond 12 months (up to 24 months), conjugates may be diluted with up to 50% glycerol and stored at -20°C to -80°C

For research requiring frequent use, consider aliquoting the antibody into single-use volumes to minimize freeze-thaw cycles and extend shelf life.

What species reactivity can be expected from TRIM3 biotin-conjugated antibodies?

The species reactivity of TRIM3 biotin-conjugated antibodies varies by manufacturer and immunogen design. Current commercial offerings show:

For the antibody described in search result , the immunogen is a synthetic peptide directed toward the N-terminal region of human TRIM3, with the sequence: TICGAKQKVLQSQLDTLRQGQEHIGSSCSFAEQALRLGSAPEVLLVRKHM .

Species predictions are typically based on sequence homology, but experimental validation is recommended when working with species not explicitly tested by the manufacturer, particularly for critical research applications.

How does biotinylation affect the complement activation properties of antibodies?

Biotinylation significantly impacts the ability of antibodies to activate the complement system. Research shows that biotinylated antibodies, including those targeting membrane proteins, are substantially weaker in causing classical complement pathway-mediated lysis of target cells compared to their non-biotinylated counterparts .

The mechanism behind this effect has been elucidated:

This property has important experimental implications:

• If complement activation is desired in your experimental system (e.g., complement-dependent cytotoxicity assays), biotinylated antibodies may be unsuitable

• Conversely, if unwanted complement activation is a concern, biotinylated antibodies provide an advantage

• In publications where both antigen recognition and complement activation need to be distinguished, biotinylated antibodies could serve as useful controls

These findings should be considered when designing experiments where complement activation might influence results.

What are the optimal methods for validating TRIM3 biotin-conjugated antibody specificity?

Rigorous validation of TRIM3 biotin-conjugated antibodies is essential for reliable experimental results. A comprehensive validation approach should include:

• Multiple detection methods:

  • Western blot: Confirm a single band of the expected molecular weight (~84 kDa for TRIM3)

  • Immunoprecipitation followed by mass spectrometry: Definitively identify the precipitated protein

  • Immunohistochemistry: Compare staining patterns with known TRIM3 expression profiles

• Genetic validation:

  • TRIM3 knockdown/knockout: Show decreased signal in cells with reduced TRIM3 expression

  • Overexpression: Demonstrate increased signal with TRIM3 overexpression

• Epitope-specific controls:

  • Peptide competition: Pre-incubate the antibody with the immunizing peptide (e.g., the N-terminal peptide used in antibody ) and show signal reduction

  • Multiple antibodies test: Compare staining patterns using antibodies targeting different TRIM3 epitopes

• Biotin-specific controls:

  • Endogenous biotin evaluation: Test streptavidin detection reagent alone on tissues

  • Biotin blocking: Compare results with and without endogenous biotin blocking

• Technical controls:

  • Isotype control: Use biotin-conjugated antibody of the same isotype but not targeting TRIM3

  • No primary antibody: Omit TRIM3 antibody but include all detection reagents

  • Cross-reactivity assessment: Test against related TRIM family proteins

Documentation of these validation steps significantly strengthens research reliability and reproducibility when working with TRIM3 biotin-conjugated antibodies.

How can biotin interference be mitigated in immunoassays using TRIM3 biotin-conjugated antibodies?

Biotin interference presents a significant challenge in assays utilizing biotin-conjugated antibodies, including those targeting TRIM3. The FDA has issued safety reports regarding biotin interference in clinical diagnostics, particularly from dietary supplements .

Sources of biotin interference:

• Endogenous biotin in biological samples (particularly serum)

• Dietary biotin supplements (can reach high concentrations in patient samples)

• Biotin-containing cell culture media components

Effective mitigation strategies include:

• Sample pretreatment:

  • Pre-absorb samples with unconjugated avidin to sequester endogenous biotin

  • Use commercial biotin blocking kits specifically designed for tissue sections or serum samples

• Assay design modifications:

  • Incorporate washing steps with biotin-free buffers

  • Add excess free biotin (e.g., 10 mM) to block unoccupied biotin-binding sites after primary detection

  • Consider alternative detection systems for highly biotin-rich samples

• Controls and validation:

  • Include biotin-spiked samples as positive controls for interference

  • Run parallel assays with non-biotin detection systems when possible

  • Implement calibration curves in biotin-containing matrices

• Technical considerations:

  • For bridged avidin-biotin (BRAB) methods, ensure complete washing to eliminate unbound biotin-labeled components

  • For labeled avidin-biotin (LAB) techniques, pre-saturate any non-specific binding sites

Implementing these strategies can significantly reduce biotin interference and improve the reliability of immunoassays using TRIM3 biotin-conjugated antibodies.

How does the degree of biotinylation impact TRIM3 antibody performance?

The extent of biotinylation (number of biotin molecules per antibody) significantly influences antibody performance, creating a balance between detection sensitivity and antigen recognition.

Optimal biotinylation typically involves 3-6 biotin molecules per antibody , but this can vary based on the specific antibody and application. The impact of biotinylation degree includes:

• Effects of over-biotinylation:

  • Reduced antigen binding if biotin molecules modify lysine residues in or near the antigen-binding site

  • Increased non-specific binding, elevating background signal

  • Altered antibody conformation affecting stability and solubility

• Effects of under-biotinylation:

  • Insufficient signal amplification

  • Reduced detection sensitivity

  • Inconsistent streptavidin binding

• Optimization recommendations:

  • When conjugating antibodies, test a range of biotin-to-antibody ratios (10-400 μg biotin per mg antibody)

  • Evaluate each conjugate through titration experiments

  • Select the conjugate with the brightest specific signal while maintaining low background

• Special considerations for TRIM3:

  • The immunogen peptide sequence for some TRIM3 antibodies contains multiple lysine residues that could be modified during biotinylation

  • If standard biotinylation affects binding, consider "long-arm" biotin (NHS-LC biotin) or carbohydrate-directed biotinylation

Commercial TRIM3 biotin-conjugated antibodies have been optimized by manufacturers, but researchers performing custom biotinylation should carefully control and validate the conjugation ratio.

Can TRIM3 biotin-conjugated antibodies be effectively used in multiplexed imaging systems?

TRIM3 biotin-conjugated antibodies can be incorporated into multiplexed imaging systems with appropriate experimental design considerations:

Advantages for multiplexing:

• Signal amplification through the biotin-streptavidin system improves detection of low-abundance proteins like TRIM3

• Various streptavidin conjugates (fluorophores, enzymes) provide detection flexibility

• Sequential staining approaches can circumvent cross-reactivity issues

Implementation strategies:

• Sequential staining protocols:

  • Use the TRIM3 biotin-conjugated antibody first

  • Detect with appropriately labeled streptavidin

  • Block remaining biotin binding sites (using free biotin at ~10 mM concentration)

  • Apply subsequent antibodies with different detection systems

• For multi-antibody panels (e.g., CITE-seq applications):

  • Pool all streptavidin-biotin antibodies together

  • Clean the pooled panel on a 50kDa cutoff column shortly before use

  • Use 1-2 μg of each antibody-biotin complex, similar to flow cytometry protocols

• Advanced multiplexing approaches:

  • Tyramide signal amplification (TSA): Use biotin-tyramide to deposit biotin at TRIM3 binding sites

  • Spectral unmixing: Utilize streptavidin conjugated to spectrally distinct fluorophores

  • Multi-round imaging: Apply antibody stripping between consecutive staining rounds

Critical controls:

• Single marker controls to establish baseline signal and bleed-through

• Full panel minus TRIM3 antibody to assess potential cross-reactivity

• Endogenous biotin control using streptavidin detection reagent alone

When properly implemented, these approaches enable effective incorporation of TRIM3 biotin-conjugated antibodies into multiplexed imaging workflows while maintaining specificity and sensitivity.

What are the recommended blocking and washing conditions when using TRIM3 biotin-conjugated antibodies?

Effective blocking and washing procedures are critical for optimizing signal-to-noise ratio when using TRIM3 biotin-conjugated antibodies. Based on research protocols and manufacturing recommendations:

Optimal blocking strategies:

• For endogenous biotin blocking:

  • Apply avidin solution (10-15 minutes)

  • Rinse briefly

  • Apply biotin solution (10-15 minutes) to block remaining avidin binding sites

  • Alternative: Use commercial biotin blocking kits optimized for tissue sections

• For general background reduction:

  • 1-5% BSA or normal serum from the same species as the secondary reagent

  • 0.1-0.3% Triton X-100 or Tween-20 to reduce non-specific hydrophobic interactions

  • Consider adding 10 mM biotin to block unoccupied biotin-binding sites in streptavidin reagents

Washing conditions:

• Buffer composition:

  • PBS with 0.05-0.1% Tween-20

  • Increased salt concentration (e.g., 0.5M NaCl) helps reduce ionic interactions

  • Avoid biotin-containing wash buffers

• Washing procedure:

  • Extend wash times (3-5 washes of 5 minutes each) after antibody incubations

  • Use gentle agitation during washing

  • Maintain consistent washing conditions between samples for reproducibility

• For complex samples:

  • Consider additional washes and higher detergent concentrations

  • Filter all reagents to remove particulates

  • For tissue sections, include a peroxidase quenching step if using HRP detection

These optimized conditions help minimize common issues such as high background, non-specific binding, and endogenous biotin interference when working with TRIM3 biotin-conjugated antibodies.

What dilution ranges are appropriate for TRIM3 biotin-conjugated antibodies in different applications?

Optimal dilution ranges for TRIM3 biotin-conjugated antibodies vary by application and specific antibody preparations. The following guidelines are based on manufacturer recommendations and standard practices:

For antibody panels (e.g., in CITE-seq):

• Use 1-2 μg of each antibody-biotin complex, comparable to flow cytometry recommendations

• Pool antibodies and clean on a 50kDa cutoff column shortly before use

Optimization recommendations:

• Perform a titration series (e.g., 1:100, 1:500, 1:1000, 1:5000) for each new lot or application

• Evaluate signal-to-noise ratio rather than absolute signal intensity

• Include positive and negative controls in titration experiments

• For custom biotinylation, test different biotin-to-antibody ratios (10-400 μg biotin per mg antibody)

Commercial TRIM3 biotin-conjugated antibodies typically come with recommended dilutions for validated applications, which should serve as starting points for optimization in specific experimental systems.

How can researchers troubleshoot non-specific binding issues with TRIM3 biotin-conjugated antibodies?

Non-specific binding is a common challenge with biotin-conjugated antibodies. When experiencing high background or unexpected staining patterns with TRIM3 biotin-conjugated antibodies, consider this systematic troubleshooting approach:

I. Identify the pattern of non-specific binding:

II. Systematic optimization strategies:

• Antibody-specific adjustments:

  • Further dilute the antibody (non-specific binding often decreases with dilution)

  • Reduce incubation time or temperature

  • Pre-absorb antibody with tissue powder from negative control samples

• Biotin/streptavidin-specific adjustments:

  • Block endogenous biotin with avidin/biotin blocking kit

  • Add free biotin (10 mM) to block unoccupied binding sites on streptavidin

  • Use streptavidin instead of avidin (less non-specific binding)

  • Filter streptavidin conjugates to remove aggregates

• Buffer and protocol modifications:

  • Increase detergent concentration in wash buffers (0.1-0.3% Triton X-100 or Tween-20)

  • Add 0.5M NaCl to reduce ionic interactions

  • Include 1-5% BSA or normal serum in diluents

  • Extend washing steps (duration and number)

• Sample preparation improvements:

  • Optimize fixation protocols (overfixation can cause high background)

  • Implement more rigorous peroxidase/phosphatase quenching for enzymatic detection

  • Test different antigen retrieval methods if applicable

These methodical approaches will help identify and resolve the specific source of non-specific binding in your experimental system when using TRIM3 biotin-conjugated antibodies.