MRPS35 Antibody

What is MRPS35 and what is its biological significance?

MRPS35 (Mitochondrial Ribosomal Protein S35) is a component of the mitochondrial ribosome responsible for protein synthesis within the mitochondrion. It is also known as 28S ribosomal protein S35 or S28mt, with a calculated molecular weight of approximately 37 kDa (323 amino acids) . Mammalian mitochondrial ribosomes (mitoribosomes) have a distinctive composition with a 4:1 ratio of protein to RNA, which differs significantly from cytoplasmic ribosomes . MRPS35 plays a critical role in the small subunit of the mitochondrial ribosome, specifically functioning as mitochondrial small ribosomal subunit protein mS35 . The protein is encoded by the MRPS35 gene (Gene ID: 60488), and its sequence information is available in databases with GenBank Accession Number BC015862 . Due to its essential role in mitochondrial translation, MRPS35 is important for understanding mitochondrial function in normal physiology and disease states.

What applications is MRPS35 Antibody validated for?

MRPS35 Antibody has been validated across multiple experimental applications, allowing for comprehensive protein analysis:

The antibody has been extensively tested with multiple positive detections in various sample types including HeLa cells, human heart tissue, and HepG2 cells for Western blot applications . For immunohistochemistry, it has been successfully used on human colon cancer and lung cancer tissue sections with appropriate antigen retrieval methods . Additionally, the antibody has been cited in at least 30 publications for Western blot applications and at least one publication for immunoprecipitation studies .

What species reactivity has been confirmed for MRPS35 Antibody?

MRPS35 Antibody demonstrates confirmed reactivity across multiple mammalian species. Most commercial preparations show reactivity with human, mouse, and rat samples . This multi-species reactivity has been experimentally validated through Western blot analysis using human cell lines (HeLa, HepG2, A549, Jurkat) and tissue lysates from both rat and mouse (heart and brain tissues) . The cross-reactivity profile makes this antibody versatile for comparative studies across species. Most cited publications have utilized this antibody for human and mouse samples . Researchers should note that while the antibody has been confirmed for these species, reactivity with other organisms has not been thoroughly evaluated and would require validation before experimental use.

What are the optimal storage conditions for MRPS35 Antibody?

To maintain antibody activity and stability, MRPS35 Antibody requires specific storage conditions. The recommended storage is at -20°C, where it remains stable for approximately one year after shipment . The antibody is typically supplied in a storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain stability during freeze-thaw cycles . For the 20μl size preparations, the solution may contain 0.1% BSA as a stabilizer . Importantly, aliquoting is generally unnecessary for -20°C storage with this preparation, which simplifies laboratory handling procedures . As with all antibodies, researchers should avoid repeated freeze-thaw cycles to preserve activity. When handling the antibody, it's recommended to briefly centrifuge the vial before opening and to maintain sterile conditions during pipetting.

How can I optimize Western blot protocols for MRPS35 detection?

Western blot optimization for MRPS35 detection requires attention to several key parameters. Based on published protocols, the following approach is recommended:

Sample preparation: Effective cell lysis is critical for MRPS35 detection. Use RIPA buffer supplemented with protease inhibitors to extract total protein from samples. Load 30 μg of protein per lane for optimal detection, as validated in experimental protocols with HeLa, HepG2, and other cell lines .

Electrophoresis conditions: Run samples on a 10% SDS-PAGE gel at 80V through the stacking gel, then increase to 120V for the resolving gel (approximately 2 hours total run time) . This provides optimal separation around the 37 kDa range, where MRPS35 is detected.

Transfer and blocking: Transfer proteins to a nitrocellulose membrane at 150 mA for 50-90 minutes, followed by blocking with 5% non-fat milk in TBS for 1.5 hours at room temperature . This reduces background and improves signal-to-noise ratio.

Antibody incubation: For primary antibody incubation, use MRPS35 antibody at 1:2000-1:10000 dilution (optimal dilution may require titration) and incubate overnight at 4°C . For enhanced specificity, wash thoroughly with TBS-0.1% Tween (three 5-minute washes) before adding HRP-conjugated secondary antibody (typically goat anti-rabbit IgG) at 1:5000 dilution for 1.5 hours at room temperature .

Expected results: A specific band should be detected at approximately 37 kDa, which corresponds to the predicted molecular weight of MRPS35 . Validation images confirm detection at this size across multiple species and sample types.

What are the recommended antigen retrieval methods for MRPS35 in immunohistochemistry?

Successful immunohistochemical detection of MRPS35 in formalin-fixed, paraffin-embedded (FFPE) tissue sections requires appropriate antigen retrieval techniques. Based on validated protocols, two primary methods have proven effective:

Heat-mediated antigen retrieval with alkaline buffer: The preferred method uses TE buffer at pH 9.0, which has been validated for optimal epitope exposure in human colon cancer tissue sections . This method effectively unmasks MRPS35 epitopes that may be cross-linked during formalin fixation.

Alternative citrate-based retrieval: For tissues that show suboptimal results with alkaline retrieval, an alternative approach using citrate buffer at pH 6.0 has also been validated . This method may be preferred for certain tissue types or fixation conditions.

For EDTA-based retrieval protocol: Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) has been successfully implemented for MRPS35 detection in both human colon cancer and lung cancer tissue sections . After retrieval, sections should be blocked with 10% goat serum to reduce non-specific binding.

The antibody concentration for IHC applications should be in the range of 1:50-1:500, with optimal dilution dependent on tissue type and fixation conditions . Incubation is typically performed overnight at 4°C, followed by appropriate secondary antibody detection systems, such as HRP-conjugated anti-rabbit IgG and DAB chromogen .

Enzyme antigen retrieval methods have also been documented for certain applications, particularly in immunocytochemical detection of MRPS35 in cell lines such as SiHa cells .

How do I troubleshoot weak or absent signal when using MRPS35 Antibody?

When encountering weak or absent signal with MRPS35 Antibody, systematic troubleshooting can identify and resolve the underlying issues:

For Western blot applications:

• Protein degradation: Ensure complete protease inhibition during sample preparation. MRPS35 is a mitochondrial protein that may be susceptible to proteolysis during cell lysis. Use fresh protease inhibitor cocktail and maintain samples on ice throughout processing .

• Inefficient transfer: MRPS35 at 37 kDa should transfer efficiently under standard conditions, but increasing transfer time (up to 90 minutes) may improve signal for problematic samples . Consider using transfer membrane with appropriate pore size (0.45 μm) for proteins in this size range.

• Antibody concentration: If signal is weak, adjust antibody concentration within the recommended range (1:2000-1:10000 for WB) . For challenging samples, try the higher end of the concentration range while monitoring background.

• Expression levels: MRPS35 expression varies across tissues and cell lines. Confirm detection in validated positive controls such as HeLa cells, HepG2 cells, or human heart tissue before concluding negative results in experimental samples .

For IHC/IF applications:

• Antigen retrieval: Inadequate antigen retrieval is a common cause of weak signal. If using TE buffer (pH 9.0) with suboptimal results, try alternative methods such as citrate buffer (pH 6.0) or EDTA buffer (pH 8.0) .

• Fixation issues: Overfixation can mask epitopes. Consider testing different fixation protocols or using freshly fixed samples with controlled fixation times.

• Antibody penetration: For tissues with dense structure, increasing antibody incubation time (up to 48 hours at 4°C) and using appropriate permeabilization agents can improve signal.

• Detection system sensitivity: If signal remains weak despite optimization, consider switching to more sensitive detection systems such as tyramide signal amplification or polymer-based detection methods.

What are the considerations for using MRPS35 Antibody in co-immunoprecipitation studies?

Co-immunoprecipitation (Co-IP) using MRPS35 Antibody requires careful optimization to maintain protein-protein interactions while achieving efficient precipitation. Based on validated protocols, consider these key factors:

Buffer composition: For studying MRPS35 interactions within the mitochondrial ribosome complex, use gentle lysis buffers that preserve native protein interactions. A recommended starting point is a buffer containing 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, and 1% NP-40 supplemented with protease inhibitors . Avoid harsh detergents like SDS that would disrupt protein-protein interactions.

Antibody amount: For optimal immunoprecipitation, use 0.5-4.0 μg of MRPS35 antibody for 1.0-3.0 mg of total protein lysate . This ratio has been validated in HeLa cells, but may require adjustment for other cell types or tissues.

Pre-clearing strategy: To reduce non-specific binding, pre-clear lysates with protein A/G beads before adding the MRPS35 antibody. This step is particularly important when working with complex samples like tissue homogenates.

Control experiments: Include appropriate controls in Co-IP experiments, such as IgG control (same host species as MRPS35 antibody) and input samples (5-10% of lysate used for IP) to validate specificity of interactions .

Elution conditions: For recovering precipitated complexes, use gentle elution with Glycine buffer (pH 2.8) followed by immediate neutralization if the goal is to preserve complex integrity for downstream applications. For direct SDS-PAGE analysis, elution with SDS-loading buffer at 95°C is sufficient.

Western blot detection: After Co-IP, validate successful precipitation by probing for MRPS35 (expected at 37 kDa) and potential interacting partners such as other mitochondrial ribosomal proteins .

Cross-linking consideration: For detecting transient or weak interactions, consider using reversible cross-linking agents like DSP (dithiobis[succinimidyl propionate]) before cell lysis to stabilize protein complexes.

How does MRPS35 expression vary across different tissues and cell lines?

MRPS35 expression demonstrates distinct patterns across tissues and cell types, reflecting its importance in mitochondrial function. Based on experimental validation data:

Tissue expression profile:

• Human heart tissue shows consistent MRPS35 expression, making it a reliable positive control for antibody validation .

• Both rat and mouse heart tissues exhibit detectable MRPS35 expression, while brain tissues from these species show comparatively lower expression levels .

• Human colon cancer tissue displays notable MRPS35 expression as validated by immunohistochemistry, suggesting potential relevance in cancer biology .

Cell line expression patterns:

• Human cell lines including HeLa, HepG2, A549, and Jurkat all demonstrate detectable MRPS35 expression in Western blot analysis, with somewhat variable expression levels .

• HeLa cells consistently show strong MRPS35 expression, making them ideal for antibody validation in multiple applications including Western blot, immunoprecipitation, and immunofluorescence .

Expression in disease states:

• Elevated MRPS35 expression has been detected in certain cancer tissues, including colon and lung cancer samples, as demonstrated by IHC validation .

• The differential expression between normal and cancer tissues suggests potential roles in cellular metabolism alterations during carcinogenesis.

Subcellular localization:

• Immunofluorescence studies in HeLa and SiHa cells confirm the expected mitochondrial localization of MRPS35, consistent with its function in mitochondrial ribosomes .

• The protein shows a characteristic punctate cytoplasmic distribution pattern typical of mitochondrial proteins.

These expression patterns should be considered when designing experiments targeting MRPS35, particularly when selecting appropriate positive controls and interpreting expression changes in experimental conditions.

What is the optimal protocol for immunofluorescence detection of MRPS35?

Immunofluorescence detection of MRPS35 requires specific methodological considerations to achieve optimal visualization of this mitochondrial ribosomal protein. Based on validated protocols:

Cell preparation and fixation:

• Culture cells on sterile coverslips to 70-80% confluence

• Fix cells with 4% paraformaldehyde in PBS for 15 minutes at room temperature

• Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes to ensure antibody access to mitochondrial targets

Antigen retrieval for immunocytochemistry:

• For certain cell types (e.g., SiHa cells), enzyme antigen retrieval may be beneficial

• Apply IHC enzyme antigen retrieval reagent for 15 minutes to enhance epitope accessibility

Blocking and antibody incubation:

• Block with 10% goat serum in PBS for 1 hour at room temperature to reduce non-specific binding

• Dilute MRPS35 antibody at 1:50-1:500 in blocking solution (optimal dilution is sample-dependent)

• Incubate with primary antibody overnight at 4°C in a humidified chamber

• Wash thoroughly with PBS (3x5 minutes)

• Apply fluorophore-conjugated secondary antibody (e.g., DyLight®488 Conjugated Goat Anti-Rabbit IgG) at 1:500 dilution for 30 minutes at 37°C

Counterstaining and mounting:

• Counterstain nuclei with DAPI (1 μg/mL) for 5 minutes

• Mount slides with anti-fade mounting medium

• Seal edges with nail polish for long-term storage

Visualization parameters:

• Observe using appropriate filter sets for the selected fluorophores

• MRPS35 typically displays a punctate cytoplasmic pattern consistent with mitochondrial localization

• Optimal exposure settings should be determined empirically to balance signal strength against background

This protocol has been successfully validated in HeLa cells (as referenced in search results) and should provide clear visualization of MRPS35 localization within cells .

How can I validate the specificity of MRPS35 Antibody for my experimental system?

Validating antibody specificity is critical for ensuring reliable experimental results. For MRPS35 Antibody, consider implementing these complementary validation approaches:

Positive and negative controls:

• Include known positive controls such as HeLa cells, HepG2 cells, or human heart tissue lysates where MRPS35 expression has been confirmed

• Use tissues with varying expression levels (e.g., heart vs. brain) to confirm signal correlation with expected expression patterns

• Consider using MRPS35 knockout or knockdown cells as negative controls to confirm signal specificity

Multiple detection methods:

• Validate across multiple applications (WB, IHC, IF) to ensure consistent detection of the target

• In Western blot, confirm that the detected band appears at the expected molecular weight (37 kDa)

• In immunofluorescence, verify subcellular localization matches the expected mitochondrial pattern

Recombinant protein competition:

• Pre-incubate the antibody with excess recombinant MRPS35 protein prior to application

• A specific antibody will show reduced or abolished signal when pre-absorbed with its target antigen

• This method is particularly valuable for validating IHC and IF applications

Orthogonal validation:

• Compare protein expression with mRNA levels using RT-PCR or RNA-seq data

• Correlation between protein and transcript levels provides additional evidence for specificity

• Consider using a second antibody targeting a different epitope of MRPS35 to confirm findings

Mass spectrometry validation:

• For definitive validation, immunoprecipitate MRPS35 using the antibody and confirm identity by mass spectrometry

• This approach can also identify potential cross-reacting proteins or novel interacting partners

For most thorough validation, combine at least three of these approaches to establish confidence in antibody specificity before proceeding with critical experiments involving MRPS35 detection.

What are the key considerations for quantitative Western blot analysis of MRPS35?

Quantitative Western blot analysis of MRPS35 requires attention to several methodological aspects to ensure accurate, reproducible results:

Sample preparation standardization:

• Maintain consistent lysis conditions across all experimental samples

• Determine protein concentration using reliable methods (BCA or Bradford assay)

• Load equal amounts of protein (30 μg recommended based on validation studies)

• Include a standard curve using recombinant MRPS35 protein for absolute quantification

Gel electrophoresis parameters:

• Use 10% SDS-PAGE gels for optimal resolution of MRPS35 (37 kDa)

• Include molecular weight markers to confirm band identity

• Consider using gradient gels (4-15%) when comparing MRPS35 with proteins of substantially different molecular weights

Transfer optimization:

• Monitor transfer efficiency using pre-stained markers

• Consider using stain-free technology or reversible membrane staining to normalize for total protein

• Transfer at 150 mA for 50-90 minutes for optimal transfer of MRPS35

Antibody dilution and incubation:

• Determine the linear detection range for your imaging system

• Optimize primary antibody concentration (1:2000-1:10000) to ensure signal falls within linear range

• Maintain consistent antibody concentration, incubation time, and temperature across experiments

Proper controls:

• Include loading controls appropriate for your experimental system

• For mitochondrial proteins like MRPS35, consider using mitochondrial markers like TOMM20 or VDAC

• Avoid housekeeping proteins that may vary under experimental conditions

Signal detection and quantification:

• Use digital imaging systems rather than film for greater dynamic range

• Capture multiple exposures to ensure signals fall within linear range

• Perform densitometry using appropriate software with background subtraction

• Normalize MRPS35 signal to total protein rather than single reference proteins for most accurate quantification

Statistical analysis:

• Run at least three biological replicates for statistical validity

• Apply appropriate statistical tests based on experimental design

• Consider using ANOVA with post-hoc tests for multi-group comparisons

These methodological considerations have been extracted from validated protocols and will help ensure reliable quantitative analysis of MRPS35 protein levels in experimental systems .

What role does MRPS35 play in mitochondrial disease research?

MRPS35 holds significant potential for mitochondrial disease research due to its fundamental role in mitochondrial translation. As a component of the small subunit of mitochondrial ribosomes, MRPS35 contributes to the synthesis of proteins encoded by mitochondrial DNA, many of which are essential components of the respiratory chain complexes . Disruptions in mitochondrial translation machinery are increasingly recognized as contributors to various mitochondrial disorders.

Research applications focusing on MRPS35 in mitochondrial disease contexts include:

• Studying MRPS35 expression alterations in patient samples with mitochondrial disorders, particularly those affecting translation. The validated immunohistochemistry protocols using MRPS35 antibody provide tools for such investigations in clinical specimens .

• Investigating potential mutations or variants in MRPS35 that might contribute to disease. Using antibodies that recognize specific regions of MRPS35 can help detect truncated or mutated forms of the protein .

• Examining the assembly and integrity of mitochondrial ribosomes in disease states through co-immunoprecipitation studies with MRPS35 antibody, which has been validated for IP applications in HeLa cells .

• Analyzing tissue-specific expression patterns of MRPS35 across affected and unaffected tissues in mitochondrial disease patients, leveraging the antibody's validated reactivity in multiple tissues including heart and cancer tissues .

• Exploring potential compensatory mechanisms in mitochondrial translation machinery during mitochondrial stress or dysfunction, using MRPS35 as a marker for small ribosomal subunit integrity.

Future directions may include developing therapeutic approaches targeting the stabilization or modulation of mitochondrial ribosomal proteins, including MRPS35, to enhance mitochondrial translation in disease states characterized by translation deficiencies.

How can MRPS35 Antibody be used in cancer research applications?

MRPS35 Antibody offers valuable applications in cancer research, as altered mitochondrial function is a hallmark of many cancers. Several specific research applications in oncology include:

Differential expression analysis:
MRPS35 Antibody has been validated for immunohistochemical detection in human colon and lung cancer tissues, allowing for comparative studies between normal and malignant tissues . This capability enables researchers to assess whether MRPS35 expression changes correlate with cancer progression, metastasis, or patient outcomes.

Cancer metabolism studies:
Mitochondrial ribosomes are crucial for synthesizing components of the oxidative phosphorylation system. Using MRPS35 Antibody in conjunction with metabolic markers can help elucidate how cancer cells modulate mitochondrial translation to support altered metabolic demands. The validated Western blot protocols across multiple cancer cell lines (HeLa, HepG2, A549, Jurkat) provide tools for such investigations .

Therapeutic response monitoring:
Cancer therapies that target mitochondrial function may affect mitochondrial translation machinery. MRPS35 Antibody can be used to assess how various treatments impact mitochondrial ribosome integrity and function in cancer models.

Subcellular localization studies:
The validated immunofluorescence protocols for MRPS35 detection enable researchers to investigate potential alterations in mitochondrial morphology or distribution in cancer cells . Combined with other mitochondrial markers, this approach can reveal cancer-specific changes in mitochondrial organization.

Biomarker development:
Given the validated detection of MRPS35 in cancer tissues, research can explore its potential as a diagnostic or prognostic biomarker, particularly in cancers where mitochondrial alterations play significant roles.

Drug discovery applications:
MRPS35 Antibody can support screening efforts for compounds that modulate mitochondrial translation in cancer cells, potentially identifying novel therapeutic approaches targeting cancer-specific metabolic vulnerabilities.

These applications leverage the validated reactivity of MRPS35 Antibody across multiple experimental techniques and sample types, providing researchers with versatile tools for investigating the complex relationships between mitochondrial translation and cancer biology .

How do I integrate MRPS35 analysis with broader mitochondrial studies?

Integrating MRPS35 analysis into comprehensive mitochondrial research requires strategic experimental design that places this ribosomal protein within the larger context of mitochondrial function. Several methodological approaches facilitate this integration:

Co-localization studies:
Utilize the validated immunofluorescence protocols for MRPS35 in combination with markers for different mitochondrial compartments (matrix, inner membrane, outer membrane) to precisely localize MRPS35 and investigate potential dynamic changes under various experimental conditions.

Proteomics integration:
Use immunoprecipitation with MRPS35 antibody coupled with mass spectrometry to identify interaction partners both within and potentially outside the mitochondrial ribosome. This approach can reveal unexpected connections between translational machinery and other mitochondrial processes.

Transcriptome-proteome correlation:
Analyze correlations between MRPS35 protein levels (detected via Western blot ) and the expression of both mitochondrial DNA-encoded and nuclear-encoded mitochondrial genes to understand regulatory relationships.

Functional consequence assessment:
After manipulating MRPS35 levels or function, measure translation rates of specific mitochondrial-encoded proteins using pulse-labeling techniques to directly link MRPS35 to its functional outcomes.

Metabolic flux analysis:
Integrate MRPS35 studies with metabolic flux analyses to determine how alterations in mitochondrial translation affect metabolic pathways dependent on mitochondrial function.

Disease model application:
Apply validated MRPS35 detection methods in disease models featuring mitochondrial dysfunction to position MRPS35 within pathological mechanisms and potential therapeutic interventions.