slc25a28 Antibody

What is SLC25A28 and why is it important in metabolic disease research?

SLC25A28, also known as mitoferrin 2, is an iron transporter located in the inner mitochondrial membrane. It plays a critical role in transporting free iron ions from the cytosol to mitochondria to support iron-dependent reactions and oxidative phosphorylation .

SLC25A28 has gained significant research attention due to its role in:

• Iron metabolism regulation

• Mitochondrial function

• Adipogenesis and obesity development

Recent studies have shown that SLC25A28 overexpression accelerates lipid accumulation in white and brown adipose tissue, enhances body weight, reduces serum triglyceride (TG) levels, and impairs glucose tolerance . This makes it a promising target for metabolic disease research, particularly in obesity and type 2 diabetes.

What are the optimal techniques for detecting SLC25A28 in different tissue types?

Based on recent research protocols, several methods have proven effective for SLC25A28 detection across different tissues:

For adipose tissue analysis, western blotting has shown excellent results in detecting SLC25A28 expression differences between white and brown adipose tissues, with brown adipose tissue showing approximately 1.5-fold higher expression after adenoviral-mediated overexpression .

What validation steps are essential before using a new SLC25A28 antibody?

A systematic validation approach should include:

• Specificity testing: Compare results using knockout/knockdown models. Recent studies have utilized SLC25A28 knockout A549 cell lysates with specific genetic modifications (10 bp deletion, 1 bp insertion, and 7 bp deletion in exon2) as negative controls .

• Cross-reactivity assessment: Test against multiple species if cross-species reactivity is claimed. Available antibodies show varying reactivity profiles (human-specific vs. multi-species reactivity) .

• Application-specific validation:

  • For Western blotting: Confirm band size (~38 kDa)

  • For immunofluorescence: Compare subcellular localization pattern with established mitochondrial markers like GRP75

  • For ELISA: Generate standard curves using recombinant protein

• Positive control selection: Use tissues known to express SLC25A28 (BAT shows higher expression levels than WAT) .

• Testing for batch consistency: Especially important when switching between lots of polyclonal antibodies .

How can SLC25A28 antibodies be optimally employed in studying the iron-obesity connection?

Recent research has uncovered a significant relationship between iron metabolism and obesity, with SLC25A28 at the intersection. For optimal investigation:

• Experimental design considerations:

  • Use paired adipose tissue and serum samples to correlate tissue SLC25A28 expression with systemic iron parameters

  • Include ATGL, perilipin 1, and adiponectin as co-investigated proteins

  • Measure both SLC25A28 expression and activity (mitochondrial iron uptake)

• Recommended protocol sequence:

  • Start with western blotting to quantify SLC25A28 expression in different adipose depots

  • Perform immunohistochemistry to visualize distribution within adipose tissue

  • Correlate expression with lipid droplet size through H&E staining

  • Measure lipid peroxidation products (MDA) to assess oxidative stress

• Critical controls:

  • Compare different metabolic states (fasting vs. fed)

  • Include both white (eWAT, iWAT) and brown adipose tissues

  • Use tissues from both standard and high-fat diet-fed animals

Research has shown that SLC25A28 overexpression in mice on high-fat diets leads to significantly increased weight gain, with adipose tissue-specific effects on proteins involved in lipogenesis and lipolysis .

What are the methodological considerations when using SLC25A28 antibodies in ferroptosis research?

Ferroptosis, an iron-dependent form of regulated cell death, has been linked to SLC25A28 function. When investigating this connection:

• Sample preparation protocols:

  • Cell lysis requires special consideration for mitochondrial membrane proteins

  • For lipid peroxidation assays, collect cells in cold PBS and process immediately to prevent ex vivo oxidation

• Critical experimental controls:

  • Include both ferroptosis inducers (RSL3) and inhibitors (DFX) with appropriate dose-response curves

  • Monitor glutathione levels alongside SLC25A28 expression

  • Measure mitochondrial complex activities (I-IV) as functional readouts

• Advanced application techniques:

  • Co-immunoprecipitation to study SLC25A28 interactions with p53 and BRD7

  • Confocal microscopy with mitochondrial markers to assess subcellular localization changes during ferroptosis

  • Detection of SLC25A28 activity through measurement of mitochondrial iron uptake using radioactive iron (55Fe)

Studies have demonstrated that the BRD7-P53-SLC25A28 axis plays a crucial role in regulating ferroptosis in hepatic stellate cells, highlighting the importance of validating both expression and activity of SLC25A28 .

How should researchers address discrepancies in SLC25A28 antibody results across different applications?

When facing contradictory results:

• Systematic troubleshooting approach:

  • Verify antibody specificity using knockout controls

  • Test multiple antibodies targeting different epitopes (N-terminal, middle region, C-terminal)

  • Compare monoclonal vs. polyclonal antibodies for the application

• Application-specific considerations:

  • For Western blotting: Membrane preparation methods significantly affect mitochondrial protein detection

  • For immunofluorescence: Fixation method impacts epitope accessibility (methanol/acetone preferable for mitochondrial proteins)

  • For flow cytometry: Permeabilization protocol critically affects detection of mitochondrial proteins

• Result interpretation framework:

  • Consider post-translational modifications that may affect epitope recognition

  • Account for potential isoforms or cleavage products

  • Evaluate potential confounding by related SLC25 family members, particularly in genetic interaction studies

When differences persist, researchers should report both results transparently, discussing potential biological or methodological explanations for the discrepancies.

What is the optimal experimental design for studying SLC25A28's role in mitochondrial function using antibody-based techniques?

Based on recent findings, a comprehensive approach should include:

• Mitochondrial isolation and fractionation:

  • Use differential centrifugation followed by density gradient separation

  • Verify fraction purity using markers for outer mitochondrial membrane, inner membrane, and matrix

• Multi-level analysis protocol:

  • Measure SLC25A28 expression by western blotting in whole cell lysates and mitochondrial fractions

  • Assess mitochondrial iron content correlation with SLC25A28 levels

  • Evaluate mitochondrial respiratory function (complexes I-IV)

  • Monitor brown adipose tissue markers (UCP-1, PGC-1α) alongside SLC25A28

• Advanced imaging approaches:

  • Super-resolution microscopy for precise subcellular localization

  • Live-cell imaging to track dynamic changes in SLC25A28 distribution

  • Co-localization with mitochondrial markers like GRP75

Research has demonstrated that SLC25A28 overexpression in brown adipose tissue leads to downregulation of UCP-1 and PGC-1α expression, suggesting a role in modulating mitochondrial biogenesis and function .

How can researchers accurately quantify SLC25A28 activity beyond expression levels?

Measuring SLC25A28 activity requires specialized approaches beyond simple protein quantification:

• Direct activity measurement:

  • Mitochondrial iron uptake assay using 55Fe-labeled substrates

  • Protocol involves incubating purified mitochondria with 55FelC3 and iron-free enterobactin, followed by quantification of mitochondrial 55Fe by liquid scintillation

• Indirect functional assessments:

  • Measure changes in mitochondrial labile iron pool

  • Assess iron-dependent enzyme activities (aconitase, complex I)

  • Monitor ROS production as a downstream effect of iron metabolism

• Integrated activity assessment framework:

  • Combine expression data (western blot/immunofluorescence) with functional readouts

  • Correlate with physiological outcomes (adipogenesis, lipid accumulation)

  • Evaluate impact on iron-dependent cellular processes

This comprehensive approach provides a more complete picture of SLC25A28 functionality beyond simple protein expression levels, which is crucial for understanding its role in disease mechanisms.

What are the best practices for using SLC25A28 antibodies in cancer research applications?

Cancer research involving SLC25A28 requires specific considerations:

• Cell line selection and validation:

  • Verify SLC25A28 expression levels across cancer cell lines using RNA-seq databases (CCLE)

  • Consider SLC25A28 knockout models for specific cancer types

  • Select appropriate control cell lines based on expression patterns

• Methodology for studying SLC25A28 in cancer metabolism:

  • Analyze co-expression with MEMO1 in cancer cell lines

  • Investigate interactions with other iron metabolism proteins

  • Correlate expression with ferroptosis sensitivity using RSL3 treatment

• Advanced analytical framework:

  • Combined analysis of SLC25A28 expression and activity with cell survival metrics

  • Investigation of SLC25A28 as a potential biomarker for treatment response

  • Correlation with other mitochondrial iron proteins

Research has indicated potential relationships between SLC25A28 function and cancer cell survival, particularly in contexts involving MEMO1 expression and iron homeostasis perturbations .

How should researchers design experiments to investigate SLC25A28's role in obesity using antibody-based techniques?

Based on recent findings, a comprehensive experimental approach should include:

• Animal model considerations:

  • Use of high-fat diet (HFD) models alongside genetic manipulation

  • Compare tissue-specific versus systemic SLC25A28 manipulation

  • Monitor body weight, adipose tissue morphology, and metabolic parameters

• Integrated analysis protocol:

  • Tissue collection from multiple adipose depots (iWAT, eWAT, BAT)

  • Histological analysis with H&E staining for adipocyte morphology

  • Protein expression analysis of lipogenesis markers (adiponectin, perilipin 1)

  • Lipolysis markers assessment (ATGL, free glycerol)

• Physiological correlations:

  • Glucose tolerance testing

  • Serum lipid profiling (TG, TC, HDL-C, LDL-C)

  • Adipokine measurements (adiponectin, FGF21, leptin)

Research has demonstrated that SLC25A28 overexpression promotes diet-induced obesity by regulating hormone secretion and inhibiting lipolysis in adipose tissue, suggesting it as a potential therapeutic target for obesity treatment .