SLC25A33 Antibody

What is SLC25A33 and what cellular functions does it perform?

SLC25A33 is a mitochondrial transporter protein that plays a critical role in importing and exporting pyrimidine nucleotides into and from mitochondria. It selectively transports uridine, thymidine, guanosine, cytosine, and inosine (deoxy)nucleoside di- and triphosphates through an antiport mechanism. This protein likely imports (deoxy)nucleoside triphosphates in exchange for intramitochondrial (deoxy)nucleoside diphosphates, thereby providing essential precursors for de novo synthesis of mitochondrial DNA and RNA while exporting products of their catabolism .

SLC25A33 has several important functions:

• Maintaining mitochondrial genome integrity

• Regulating mitochondrial membrane potential

• Supporting mitochondrial respiration

• Controlling mitochondrial DNA replication and transcription in response to insulin (INS) or insulin-like growth factor 1 (IGF1) stimulation

• Regulating cell growth and proliferation by influencing the ratio of mitochondria-to-nuclear-encoded components of the electron transport chain

• Modulating mitochondrial ROS production

• Participating in dendritic cell endocytosis

The protein is also known by several alternative names: Bone marrow stromal cell mitochondrial carrier protein, Protein PNC1, BMSC-MCP, HuBMSC-MCP, and MGC4399 .

What types of SLC25A33 antibodies are commercially available?

There is a wide range of SLC25A33 antibodies available from various manufacturers. According to the Antibodypedia database, there are approximately 77 antibodies from 19 different providers . The most common type is polyclonal antibodies, though monoclonal options may also be available.

Some of the top validated antibodies include:

• ABIN442467 from antibodies-online (Polyclonal, suitable for WB and IHC)

• GTX106785 from GeneTex (Polyclonal, suitable for WB and IHC)

• LS-C185836 from LSBio (Polyclonal, suitable for WB and IHC)

• NBP1-32499 from Novus Biologicals (Polyclonal, suitable for WB and IHC)

• 17794-1-AP from Proteintech Group (Polyclonal, suitable for WB and ELISA)

When selecting an antibody, researchers should consider those with validation data and references supporting their specificity and performance in the intended application.

What applications are SLC25A33 antibodies validated for?

Based on the available information, SLC25A33 antibodies have been primarily validated for the following applications:

• Western Blotting (WB): This is the most commonly validated application, with multiple antibodies showing successful detection of SLC25A33 in human samples and specific cell lines like U2Os .

• Immunohistochemistry (IHC): Several antibodies are reported to work for tissue staining, allowing visualization of SLC25A33 expression patterns in different tissues .

• ELISA (Enzyme-Linked Immunosorbent Assay): At least one antibody (17794-1-AP from Proteintech) has been validated for ELISA applications .

It's important to note that not all antibodies are validated for all applications, and researchers should select antibodies specifically validated for their intended experimental approach.

What are the recommended protocols for using SLC25A33 antibodies in Western blotting?

For optimal results when using SLC25A33 antibodies in Western blotting, consider the following protocol recommendations:

Sample Preparation:

• Extract proteins using a lysis buffer that preserves mitochondrial proteins (typically containing detergents like NP-40 or Triton X-100)

• Include protease inhibitors to prevent degradation

• For mitochondrial proteins like SLC25A33, consider using mitochondrial isolation protocols if studying subcellular localization

Electrophoresis Conditions:

• Use 10-12% SDS-PAGE gels for optimal separation

• Load adequate protein amount (typically 20-40 μg of total protein per lane)

• Include molecular weight markers spanning the expected size range of SLC25A33 (~35 kDa)

Transfer and Detection:

• Use PVDF membranes for better protein retention

• Block with 5% non-fat milk or BSA in TBST

• For primary antibody incubation, dilute according to manufacturer's recommendations (typically 1:500-1:2000)

• Incubate overnight at 4°C for optimal binding

• Use appropriate HRP-conjugated secondary antibodies

Positive Controls:

• U2Os cells have been confirmed to express detectable levels of SLC25A33 and can serve as a positive control

Expected Results:

• The expected molecular weight of SLC25A33 is approximately 35 kDa

• Validation might include detection of recombinant SLC25A33 protein alongside endogenous protein

Following these guidelines should help ensure specific detection of SLC25A33 in Western blot experiments.

How can researchers validate the specificity of SLC25A33 antibodies?

Validating antibody specificity is crucial for ensuring reliable research results. For SLC25A33 antibodies, consider implementing these validation approaches:

Positive and Negative Controls:

• Use cell lines with confirmed SLC25A33 expression (e.g., U2Os cells) as positive controls

• Compare with cell lines where SLC25A33 expression is absent or knocked down

• Include recombinant SLC25A33 protein as a positive control

Knockdown/Knockout Validation:

• Perform siRNA knockdown or CRISPR-Cas9 knockout of SLC25A33

• Compare antibody staining/detection between wild-type and knockdown/knockout samples

• Observe reduction or disappearance of the target band/signal

Peptide Competition Assay:

• Pre-incubate the antibody with excess immunizing peptide

• This should block specific antibody binding and reduce or eliminate specific signals

• Non-specific signals will remain unaffected

Orthogonal Method Validation:

• Correlate protein detection with mRNA expression (RT-qPCR)

• Confirm subcellular localization matches expected mitochondrial pattern

• Use multiple antibodies targeting different epitopes of SLC25A33

Mass Spectrometry Confirmation:

• Perform immunoprecipitation with the antibody

• Analyze the precipitated proteins by mass spectrometry

• Confirm the presence of SLC25A33 peptides

This multi-faceted approach provides robust validation of antibody specificity, ensuring confidence in experimental results.

What controls should be included in experiments using SLC25A33 antibodies?

Including appropriate controls is essential for interpreting results obtained with SLC25A33 antibodies:

Positive Controls:

• Cell lines with confirmed SLC25A33 expression (e.g., U2Os cells)

• Tissues known to express SLC25A33 (based on RNA-seq data)

• Recombinant SLC25A33 protein (especially useful for Western blot)

Negative Controls:

• Cell lines with minimal or no SLC25A33 expression

• SLC25A33 knockdown or knockout samples

• Secondary antibody-only controls to assess non-specific binding

• Isotype control antibodies to identify Fc-mediated binding

Additional Technical Controls:

• Peptide competition controls (pre-incubation with immunizing peptide)

• Loading controls for Western blot (β-actin, GAPDH, or mitochondrial markers)

• For IHC, include no-primary-antibody controls on adjacent tissue sections

By including these controls, researchers can confidently interpret their findings and troubleshoot any unexpected results.

How can SLC25A33 antibodies be used to study mitochondrial nucleotide transport?

SLC25A33 plays a critical role in mitochondrial nucleotide transport, which is essential for mitochondrial DNA and RNA synthesis. Researchers can leverage SLC25A33 antibodies to advance understanding in this area through several approaches:

Co-localization Studies:

• Use SLC25A33 antibodies in immunofluorescence experiments alongside mitochondrial markers

• Visualize the distribution of SLC25A33 within the mitochondrial network

• Examine potential redistribution under different metabolic conditions

Protein-Protein Interaction Studies:

• Perform co-immunoprecipitation using SLC25A33 antibodies to identify interacting partners

• Use proximity ligation assays to visualize interactions in situ

• Combine with crosslinking approaches to capture transient interactions

Functional Assays:

• Monitor nucleotide transport in isolated mitochondria with and without SLC25A33 inhibition

• Correlate SLC25A33 expression levels (detected by antibodies) with nucleotide transport efficiency

• Examine changes in mitochondrial DNA synthesis rates in relation to SLC25A33 expression

Response to Cellular Signals:

• Analyze SLC25A33 expression and localization changes in response to insulin or IGF1 stimulation

• Compare these changes with alterations in mitochondrial DNA replication and transcription

• Investigate the role of SLC25A33 in modulating mitochondrial ROS production

What role does SLC25A33 play in cell growth and proliferation, and how can antibodies help investigate this?

SLC25A33 has been implicated in regulating cell growth and proliferation, particularly in response to insulin (INS) or insulin-like growth factor 1 (IGF1) stimulation . Researchers can use SLC25A33 antibodies to investigate these functions through several methodological approaches:

Signaling Pathway Analysis:

• Examine SLC25A33 expression changes in response to growth factors using Western blot

• Correlate changes in SLC25A33 levels with activation of growth-related signaling pathways

• Investigate post-translational modifications of SLC25A33 that might regulate its activity

Cell Cycle Studies:

• Analyze SLC25A33 expression and localization throughout different phases of the cell cycle

• Compare SLC25A33 levels between quiescent and proliferating cells

• Investigate the impact of SLC25A33 knockdown on cell cycle progression

Mitochondrial Function Assessment:

• Measure parameters like mitochondrial membrane potential and respiration in relation to SLC25A33 levels

• Analyze the ratio of mitochondria-to-nuclear-encoded components of the electron transport chain

• Evaluate mitochondrial ROS production in cells with different SLC25A33 expression levels

Cancer Research Applications:

• Compare SLC25A33 expression between normal and cancer cells

• Investigate whether SLC25A33 contributes to the metabolic reprogramming observed in cancer cells

• Explore the potential of SLC25A33 as a therapeutic target or biomarker

By utilizing SLC25A33 antibodies in these research contexts, investigators can gain deeper insights into how this mitochondrial transporter influences cellular growth and proliferation.

What are common issues when working with SLC25A33 antibodies and how can they be resolved?

When working with SLC25A33 antibodies, researchers may encounter several common challenges. Here are troubleshooting approaches for addressing these issues:

Weak or Absent Signal in Western Blot:

• Increase protein loading (try 40-60 μg of total protein)

• Optimize antibody concentration (try a titration series)

• Increase incubation time (overnight at 4°C)

• Use more sensitive detection systems (enhanced chemiluminescence or fluorescent secondaries)

• Consider sample preparation methods that better preserve mitochondrial proteins

• Use U2Os cells as a positive control to confirm antibody functionality

High Background:

• Increase blocking time or concentration (5% milk or BSA)

• Use more stringent washing conditions (increase wash duration and number of washes)

• Reduce secondary antibody concentration

• Pre-absorb antibody against common cross-reactive proteins

• Try alternative blocking agents (casein, fish gelatin)

Non-specific Bands:

• Confirm the expected molecular weight of SLC25A33 (~35 kDa)

• Perform peptide competition assays to identify specific bands

• Use gradient gels for better separation

• Try more specific lysis buffers to reduce proteolysis

• Consider using fresh samples to minimize degradation

Poor Reproducibility:

• Standardize protein extraction and quantification methods

• Maintain consistent antibody lots when possible

• Document exact experimental conditions for replication

• Consider the effects of cell confluency and passage number on expression levels

By systematically addressing these common issues, researchers can optimize their experiments with SLC25A33 antibodies and obtain more reliable and reproducible results.

How can researchers optimize immunohistochemistry protocols for SLC25A33 detection?

Optimizing immunohistochemistry (IHC) protocols for SLC25A33 detection requires careful attention to several key parameters:

Antigen Retrieval Optimization:

• Test multiple antigen retrieval methods (heat-induced epitope retrieval using citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Optimize retrieval duration and temperature

• For mitochondrial proteins like SLC25A33, heat-mediated retrieval is often more effective

Fixation Considerations:

• Compare different fixatives (4% paraformaldehyde, formalin, methanol)

• Optimize fixation duration to preserve epitope accessibility

• For frozen sections, test post-fixation protocols

Antibody Dilution Series:

• Perform a dilution series to determine optimal antibody concentration

• Typical starting ranges for SLC25A33 antibodies in IHC are 1:50-1:200

• Extend incubation times (overnight at 4°C) for weaker antibodies

Detection System Selection:

• Compare different detection systems (ABC, polymer-based)

• For low abundance proteins like SLC25A33, amplification systems may improve sensitivity

• Consider tyramide signal amplification for enhanced detection

Background Reduction Strategies:

• Block endogenous peroxidase activity thoroughly

• Use species-specific blocking agents

• Include additional blocking steps with BSA or normal serum

• Add detergents (0.1-0.3% Triton X-100) to reduce non-specific binding

Counterstaining Optimization:

• Adjust counterstain intensity to ensure visibility of SLC25A33 staining

• For co-localization studies, select compatible fluorophores with minimal spectral overlap

By systematically optimizing these parameters, researchers can develop robust IHC protocols for specific detection of SLC25A33 in tissue samples.

How are SLC25A33 antibodies being used in current mitochondrial research?

SLC25A33 antibodies are valuable tools in cutting-edge mitochondrial research, facilitating investigations into several key areas:

Mitochondrial Genome Maintenance:

• Researchers use SLC25A33 antibodies to study its role in maintaining mitochondrial DNA integrity

• Investigations focus on how nucleotide transport affects mitochondrial genome replication fidelity

• Studies examine the correlation between SLC25A33 expression and mitochondrial DNA copy number

Mitochondrial Dysfunction in Disease:

• SLC25A33 antibodies help investigate alterations in expression or localization in disease states

• Studies explore the role of SLC25A33 in neurodegenerative diseases with mitochondrial involvement

• Research examines potential contributions to metabolic disorders through altered nucleotide transport

Mitochondrial Response to Cellular Signaling:

• Antibodies enable visualization of SLC25A33 regulation in response to insulin and IGF1 signaling

• Investigations probe how these signals translate to changes in mitochondrial function

• Studies analyze how SLC25A33-mediated nucleotide transport affects mitochondrial ROS production

Mitochondrial-Nuclear Communication:

• Research explores SLC25A33's role in coordinating mitochondrial and nuclear genome expression

• Studies examine how the ratio of mitochondria-to-nuclear-encoded components is maintained

• Investigations focus on retrograde signaling mechanisms that may involve SLC25A33

These applications highlight the versatility of SLC25A33 antibodies in advancing our understanding of fundamental mitochondrial biology and its implications for health and disease.

What are promising future directions for research involving SLC25A33 antibodies?

Several promising research directions could leverage SLC25A33 antibodies to advance scientific understanding:

Single-Cell Analysis:

• Developing protocols for single-cell immunofluorescence to analyze SLC25A33 expression heterogeneity

• Correlating SLC25A33 levels with mitochondrial function at the single-cell level

• Examining cell-to-cell variability in SLC25A33 expression within tissues

Dynamic Regulation Studies:

• Using live-cell imaging with fluorescently tagged antibody fragments to track SLC25A33 dynamics

• Investigating real-time changes in SLC25A33 localization in response to metabolic challenges

• Studying the kinetics of SLC25A33 turnover under different cellular conditions

Therapeutic Target Validation:

• Utilizing SLC25A33 antibodies to validate it as a potential therapeutic target in diseases with mitochondrial dysfunction

• Screening compounds that modulate SLC25A33 function using antibody-based assays

• Developing antibody-drug conjugates for targeted delivery to cells with aberrant SLC25A33 expression

Multi-omics Integration:

• Combining antibody-based detection with proteomics and transcriptomics

• Correlating SLC25A33 protein levels with global changes in mitochondrial proteome

• Integrating data from antibody-based assays with metabolomic profiles to understand functional consequences

Biosensor Development:

• Creating antibody-based biosensors for real-time monitoring of SLC25A33 function

• Developing split-fluorescent protein systems incorporating SLC25A33 antibody fragments

• Engineering FRET-based sensors to detect SLC25A33 conformational changes during transport

These future directions represent exciting opportunities for researchers to leverage SLC25A33 antibodies in advancing our understanding of mitochondrial biology and developing novel therapeutic approaches.

What are the key considerations for selecting and using SLC25A33 antibodies?

When selecting and using SLC25A33 antibodies for research applications, consider these key recommendations:

• Validation Status: Choose antibodies with published validation data and references demonstrating specificity for SLC25A33 .

• Application Suitability: Select antibodies specifically validated for your intended application (WB, IHC, ELISA) .

• Species Reactivity: Ensure the antibody has been validated for your species of interest, with human reactivity being the most commonly validated .

• Controls: Always include appropriate positive controls (such as U2Os cells) and negative controls in your experiments .

• Protocol Optimization: Be prepared to optimize protocols for your specific experimental conditions, particularly for techniques like IHC where tissue preparation can significantly impact results.

• Batch Consistency: When possible, use the same antibody lot for related experiments to minimize variability.

• Storage and Handling: Follow manufacturer recommendations for storage conditions and avoid repeated freeze-thaw cycles to maintain antibody integrity.

• Complementary Approaches: Consider using multiple antibodies targeting different epitopes of SLC25A33 to increase confidence in your findings.

By carefully considering these factors, researchers can maximize the reliability and reproducibility of their experiments involving SLC25A33 antibodies.

How can researchers contribute to improving SLC25A33 antibody validation standards?

Researchers can play an important role in advancing antibody validation standards for SLC25A33 and other targets through these approaches:

• Comprehensive Reporting: Document detailed protocols, including specific antibody information (catalog number, lot, dilution) in publications.

• Validation Data Sharing: Share antibody validation data in publications and repositories, including both positive and negative results.

• Multi-application Validation: Test and report antibody performance across multiple applications to expand known utility.

• Knockout Validation: When possible, validate antibodies using genetic approaches like CRISPR-Cas9 knockout of SLC25A33.

• Community Engagement: Participate in antibody validation initiatives and contribute to databases like Antibodypedia .

• Reproducibility Studies: Conduct and publish studies replicating previous findings with independent antibody lots or sources.

• Methodological Innovation: Develop and share new approaches for antibody validation specific to mitochondrial proteins like SLC25A33.

• Cross-laboratory Validation: Collaborate with other research groups to validate antibody performance across different laboratory settings.