PRELID3B Antibody

What is PRELID3B and what cellular functions does it perform?

PRELID3B (also known as SLMO2, C20orf45, Protein slowmo homolog 2) is a 21kDa protein that plays a crucial role in mitochondrial lipid homeostasis . The protein is predicted to enable phosphatidic acid transfer activity and is involved in phospholipid transport within cells . Research indicates that PRELID3B is primarily active in the mitochondrial intermembrane space, suggesting its importance in maintaining proper mitochondrial function .

While earlier studies indicated that the function of this protein remained unknown , more recent research has connected PRELID3B to important roles in cellular lipid metabolism. Understanding its function is critical for researchers aiming to investigate mitochondrial lipid transport mechanisms or disorders related to mitochondrial dysfunction.

For maximum stability and activity retention, PRELID3B antibodies should be stored at -20°C, avoiding repeated freeze-thaw cycles that can degrade antibody performance . Most commercial preparations are supplied in a phosphate-buffered solution (pH 7.3-7.4) containing stabilizers and 50% glycerol to prevent freeze damage .

When working with these antibodies:

• Aliquot the stock solution upon first thawing to minimize freeze-thaw cycles

• Store working dilutions at 4°C for short-term use (1-2 weeks maximum)

• Return stock solutions immediately to -20°C after use

• Monitor storage conditions carefully as temperature fluctuations can significantly impact antibody binding efficiency and specificity

Most manufacturers validate stability for 12 months when stored under recommended conditions .

What are the optimal blocking conditions for Western blotting with PRELID3B antibodies?

When performing Western blotting with PRELID3B antibodies, optimal blocking conditions are critical for reducing background and enhancing specific signal detection. Validated protocols recommend using 3-5% nonfat dry milk in TBST as an effective blocking buffer . This blocking approach helps minimize non-specific binding while preserving specific antibody-antigen interactions.

For the detection procedure:

• Block membranes in 3% nonfat dry milk in TBST for 1 hour at room temperature

• Incubate with primary PRELID3B antibody at dilutions ranging from 1:500 to 1:1000

• Use HRP-conjugated secondary antibodies (such as Goat Anti-Rabbit IgG) at approximately 1:10,000 dilution

• Detect signal using standard ECL reagents with exposure times starting at 60 seconds

For challenging samples or when background issues persist, researchers may alternatively test 5% BSA in TBST as a blocking agent, particularly when phospho-specific interactions might be affected by components in milk-based blockers.

How can researchers validate the specificity of PRELID3B antibodies in their experimental systems?

Validating antibody specificity is essential for generating reliable research data. For PRELID3B antibodies, a multi-faceted validation approach is recommended:

• Molecular weight verification: Confirm detection of the expected 21kDa band by Western blot, consistent with the calculated molecular weight of PRELID3B

• Positive control selection: Use validated cell lines known to express PRELID3B, such as 293T cells, which have been documented as reliable positive controls

• Knockdown/knockout validation: Implement siRNA knockdown or CRISPR-Cas9 knockout of PRELID3B, comparing antibody signals between control and depleted samples

• Recombinant protein controls: Use purified recombinant PRELID3B protein (available as full-length 1-194aa constructs) for competition assays or as positive controls

• Cross-reactivity testing: When working with non-human samples, validate species cross-reactivity experimentally even when manufacturers claim reactivity, as performance can vary across species

This comprehensive validation approach ensures that experimental findings accurately reflect PRELID3B biology rather than artifacts from non-specific antibody interactions.

What tissue samples have been successfully used for immunohistochemistry with PRELID3B antibodies?

Immunohistochemical detection of PRELID3B has been successfully demonstrated in several human tissue types, with particular success in:

• Human liver cancer tissue

• Human colorectal cancer tissue

When performing IHC with PRELID3B antibodies:

• Use recommended dilutions of 1:100-1:300 for optimal staining

• Include positive control tissues in each experimental run

• Implement appropriate antigen retrieval methods (detailed protocols typically provided by manufacturers)

• Consider parallel staining with different antibody clones to confirm staining patterns

Researchers investigating PRELID3B distribution in normal tissues should first establish staining conditions using these validated tissue types before extending to other specimens. This methodical approach helps establish reliable baseline staining parameters for subsequent experiments.

How can PRELID3B antibodies be used to investigate mitochondrial lipid transport mechanisms?

Given PRELID3B's predicted function in phosphatidic acid transfer and mitochondrial lipid homeostasis , antibodies against this protein can serve as valuable tools for investigating these critical cellular processes:

• Co-localization studies: Combine PRELID3B antibodies with mitochondrial markers in immunofluorescence microscopy to analyze its precise sub-mitochondrial localization. Focus particularly on the intermembrane space where the protein is predicted to function .

• Protein complex identification: Utilize PRELID3B antibodies for co-immunoprecipitation experiments to identify interaction partners involved in lipid transport machinery.

• Lipid transfer assays: Develop in vitro assays where PRELID3B function is monitored before and after immunodepletion with specific antibodies, measuring changes in phospholipid transfer between membrane fractions.

• Stress response analysis: Monitor PRELID3B localization and expression changes using validated antibodies during conditions that challenge mitochondrial lipid homeostasis, such as oxidative stress or lipid imbalance.

The methodological approach should include careful subcellular fractionation to separate mitochondrial components, followed by Western blotting or immunofluorescence with PRELID3B antibodies to track protein distribution and abundance under various experimental conditions.

What considerations should be made when using different detection systems with PRELID3B antibodies?

Different detection systems can significantly impact the sensitivity and specificity of PRELID3B antibody-based assays. Researchers should consider these methodological factors:

For fluorescence-based detection:

• Select fluorophores with emission spectra appropriate for the subcellular localization of PRELID3B to avoid autofluorescence from mitochondria

• Consider photobleaching characteristics during prolonged imaging of mitochondrial structures

• Implement appropriate controls to distinguish specific PRELID3B signals from mitochondrial autofluorescence

For enzymatic detection systems (HRP/AP):

• ECL Basic Kit detection systems have been validated for PRELID3B Western blotting applications

• Exposure times of approximately 60 seconds have been demonstrated to produce clear signals when using HRP-conjugated secondary antibodies at 1:10,000 dilution

• For low-abundance samples, consider enhanced chemiluminescence substrates or signal amplification systems

The selection of detection methodology should be guided by the experimental question, with consideration given to the relative abundance of PRELID3B in the target tissues and the spatial resolution required for the analysis.

What are common issues in Western blotting with PRELID3B antibodies and how can they be resolved?

Western blotting for PRELID3B may encounter several technical challenges that can be systematically addressed:

For optimal results with PRELID3B Western blotting:

• Use freshly prepared lysates when possible

• Include protease inhibitors during sample preparation

• Optimize exposure times, starting with the validated 60-second exposure and adjusting as needed

These methodological adjustments address the most common technical barriers while maintaining experimental rigor.

How can researchers optimize immunohistochemistry protocols for PRELID3B detection in difficult tissue samples?

When working with challenging tissue samples for PRELID3B immunohistochemistry, several optimization strategies can enhance detection specificity and sensitivity:

• Antigen retrieval optimization:

  • Test multiple antigen retrieval methods (heat-induced epitope retrieval in citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Adjust retrieval duration in 5-minute increments to identify optimal conditions

  • For formalin-fixed tissues, extended retrieval times may be necessary to unmask epitopes

• Signal amplification systems:

  • Implement tyramide signal amplification when working with tissues exhibiting low PRELID3B expression

  • Consider polymer-based detection systems that provide enhanced sensitivity without increasing background

• Background reduction:

  • Pre-absorb primary antibodies with tissue homogenates from species of origin

  • Include additional blocking steps with species-specific normal serum

  • Reduce endogenous peroxidase activity with extended hydrogen peroxide incubation

• Controls and validation:

  • Include verified positive control tissues (human liver cancer or colorectal cancer)

  • Implement absorption controls using the immunizing peptide when available

  • Use serial dilutions to identify optimal antibody concentration for each tissue type

These methodological refinements should be systematically implemented and documented to establish reproducible protocols for specific tissue types.

What strategies can address cross-reactivity concerns when using PRELID3B antibodies in comparative species studies?

When conducting comparative studies across species using PRELID3B antibodies, addressing potential cross-reactivity issues is critical for data interpretation:

• Sequence homology analysis:

  • Perform alignment analysis of PRELID3B sequences across target species

  • Focus particularly on the region corresponding to the immunogen (amino acids 80-180 in human PRELID3B)

  • Predict potential epitope conservation based on sequence identity percentages

• Validation in each species:

  • Perform Western blot analysis in tissues from each target species

  • Verify the molecular weight of detected proteins matches the predicted PRELID3B size in that species

  • Include positive control samples with confirmed PRELID3B expression

• Knockout/knockdown controls:

  • Where possible, use species-specific PRELID3B knockout or knockdown samples as negative controls

  • These controls are particularly valuable when antibodies claim broad cross-reactivity across species

• Alternative antibody selection:

  • Consider using multiple antibodies raised against different epitopes

  • Select antibodies specifically validated in your species of interest

  • For evolutionary studies, prioritize antibodies generated against highly conserved regions

How can PRELID3B antibodies contribute to understanding mitochondrial dysfunction in disease models?

PRELID3B antibodies offer valuable tools for investigating mitochondrial lipid homeostasis disruptions in various disease contexts:

• Neurodegenerative disease models:

  • Track PRELID3B expression and localization changes in cell and animal models of neurodegeneration

  • Correlate alterations with mitochondrial function parameters and lipid composition

  • Investigate potential therapeutic interventions targeting PRELID3B pathways

• Cancer metabolism studies:

  • Examine PRELID3B expression in cancer tissues where mitochondrial metabolism is altered

  • Investigate connections between PRELID3B levels and metabolic reprogramming in tumors

  • Explore potential diagnostic applications in conjunction with other mitochondrial markers

• Cardiac and metabolic disorders:

  • Analyze PRELID3B distribution in tissues affected by ischemia-reperfusion injury

  • Examine changes during metabolic syndrome progression

  • Correlate PRELID3B alterations with cardiac mitochondrial function

• Aging research:

  • Monitor age-dependent changes in PRELID3B expression and mitochondrial localization

  • Investigate potential interventions that normalize PRELID3B function in aged tissues

These research directions leverage antibody-based detection methods to build mechanistic understandings of how PRELID3B contributes to maintaining mitochondrial health across diverse physiological and pathological states.

What emerging techniques might enhance the utility of PRELID3B antibodies in future research?

Several cutting-edge methodological approaches hold promise for expanding the research applications of PRELID3B antibodies:

• Super-resolution microscopy:

  • Apply techniques like STORM, PALM, or STED microscopy with PRELID3B antibodies

  • Resolve sub-mitochondrial localization with precision beyond conventional microscopy

  • Map spatial relationships between PRELID3B and other components of lipid transport machinery

• Proximity labeling approaches:

  • Combine PRELID3B antibodies with BioID or APEX2 proximity labeling

  • Identify proteins in close physical proximity to PRELID3B within mitochondrial membranes

  • Generate comprehensive interaction networks under different physiological conditions

• Live-cell imaging adaptations:

  • Develop membrane-permeable antibody fragments targeting PRELID3B

  • Create nanobody alternatives for live-cell tracking of PRELID3B dynamics

  • Implement FRET-based reporters to monitor PRELID3B interactions in real-time

• Mass spectrometry integration:

  • Use PRELID3B antibodies for immunoprecipitation followed by mass spectrometry

  • Identify post-translational modifications and interaction partners

  • Quantify changes in the PRELID3B interactome under different conditions

These methodological advances will likely transform how researchers utilize PRELID3B antibodies, moving beyond static detection toward dynamic analyses of protein function in complex cellular environments.