FASTKD2 Antibody, HRP conjugated

What is FASTKD2 and what is its cellular localization?

FASTKD2 (FAST kinase domain-containing protein 2) is a member of the human FASTK protein family, which includes six architecturally related proteins (FASTK and FASTKD1-5) that localize to mitochondria . Specifically, FASTKD2 is located in the mitochondrial inner compartment. Experimental evidence confirms this localization through immunofluorescence studies using HA-tagged FASTKD2 constructs transfected into COS7 and HeLa cells, where the FASTKD2-specific immunofluorescence pattern coincided with that of mitochondrial single-strand-DNA binding protein (mtSSB), a mitochondrion-specific protein . FASTKD2 contains a FAST kinase domain and two predicted transmembrane domains (amino acid residues 403-421 and 482-500) .

What are the typical applications for FASTKD2 antibodies in research?

FASTKD2 antibodies can be utilized in multiple experimental applications as demonstrated by published research:

These applications allow researchers to investigate FASTKD2 expression, localization, binding partners, and functional roles in mitochondrial RNA metabolism .

What is the significance of the discrepancy between predicted and observed molecular weights of FASTKD2?

The calculated molecular weight of FASTKD2 is 81 kDa (710 amino acids), yet the observed molecular weight in Western blots is typically 55-65 kDa . This discrepancy may be explained by alternative translation initiation. Research suggests that the "true" FASTKD2 protein likely starts from methionine at position 17 (M17) rather than position 1 (M1), as supported by sequence conservation analysis across species . Additionally, mitochondrial-targeting prediction software shows higher scores for mitochondrial targeting with the sequence starting at M17 compared to M1 (Mitoprot: 91% versus 72%; TargetP: 76% versus 43%) . This knowledge is crucial for researchers to correctly interpret Western blot results and design FASTKD2-targeting constructs.

What is the tissue distribution pattern of FASTKD2 expression?

FASTKD2 is ubiquitously expressed but shows variable expression levels across different tissues. Experimental analysis using real-time PCR of mouse tissues revealed a distinct expression pattern: skeletal muscle < heart ≤ kidney < liver ≤ brain . This tissue-specific expression pattern may correlate with the varying mitochondrial content and activity in these tissues. Researchers should consider these expression differences when designing experiments targeting endogenous FASTKD2 in different tissue types or cell lines.

How should FASTKD2 antibodies be stored and handled to maintain their efficacy?

FASTKD2 antibodies, particularly HRP-conjugated versions, require specific storage conditions to maintain their activity. The recommended storage parameters are:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles

• For longer-term storage, consider aliquoting the antibody (although 20μl sizes containing 0.1% BSA may not require aliquoting for -20°C storage)

• Typical storage buffer consists of PBS with 0.02-0.03% preservative (such as Proclin 300), 50% glycerol, pH 7.4

Proper handling ensures optimal antibody performance in all applications, particularly for sensitive techniques like immunofluorescence where signal-to-noise ratio is critical.

How can FASTKD2 antibodies be used to study mitochondrial RNA processing mechanisms?

FASTKD2 plays a crucial role in mitochondrial RNA processing and is part of a protein complex involved in pseudouridylation of mitochondrial RNA . To investigate these mechanisms, researchers can employ:

• RNA Immunoprecipitation (RIP): Using FASTKD2 antibodies to pull down protein-RNA complexes, followed by RNA sequencing to identify FASTKD2-bound transcripts. This approach can reveal which mitochondrial RNAs directly interact with FASTKD2.

• Co-immunoprecipitation (Co-IP): Using FASTKD2 antibodies to identify protein interaction partners such as RPUSD3, RPUSD4, NGRN, WBSCR16, and PTCD1 .

• CLIP-seq approaches: For high-resolution mapping of FASTKD2-RNA interactions in vivo.

• Immunofluorescence co-localization: With mitochondrial RNA granule markers to investigate FASTKD2's involvement in RNA granule formation and function.

These approaches can help elucidate FASTKD2's specific role in post-transcriptional regulation of mitochondrial gene expression, particularly in RNA maturation processes.

How do FASTKD2 knockdown or knockout models affect mitochondrial transcriptome and function?

Studies with FASTKD2-deficient models have revealed important insights into its function. While specific knockout data for FASTKD2 is limited in the provided search results, research on related family members indicates distinct effects on mitochondrial RNA processing .

Unlike some family members (FASTKD3, FASTKD4, and FASTKD5), whose knockout produces specific processing defects at canonical and non-canonical RNA junctions, FASTKD2's precise role appears more specialized. Mutations in FASTKD2 have been associated with mitochondrial encephalomyopathy and cytochrome c oxidase (COX) deficiency in skeletal muscle .

Interestingly, blue-native gel electrophoresis (BNGE) analysis of patient muscle lacking functional FASTKD2 did not show abnormalities in COX assembly, suggesting that the COX defect might be part of a broader mitochondrial dysfunction rather than a direct assembly defect . This indicates FASTKD2 may have a distinct function from other family members in mitochondrial RNA metabolism.

What are the optimal experimental conditions for immunoprecipitation using FASTKD2 antibodies?

For successful immunoprecipitation with FASTKD2 antibodies, the following protocol parameters have been validated:

• Antibody amount: 0.5-4.0 μg for every 1.0-3.0 mg of total protein lysate

• Validated cell types: HepG2 cells show positive results for IP

• Lysis conditions: Standard IP lysis buffers containing mild detergents (NP-40 or Triton X-100) are suitable

• Binding conditions: Overnight incubation at 4°C with gentle rotation

• Washing steps: Multiple washes with decreasing detergent concentrations

• Elution: Either by boiling in SDS sample buffer for Western blot analysis or gentle elution for maintaining protein complexes

For RNA immunoprecipitation (RIP) applications, RNase inhibitors must be included in all buffers, and additional crosslinking steps may be necessary to preserve transient RNA-protein interactions.

How can researchers validate the specificity of FASTKD2 antibodies in their experimental system?

Validating antibody specificity is crucial for reliable results. For FASTKD2 antibodies, consider these validation approaches:

• Positive and negative control samples: Use cell lines with known FASTKD2 expression (HeLa, HepG2, K-562 are positive controls) . Note that HAP1 cells lack FASTKD2 expression and can serve as a negative control .

• Knockdown/knockout validation: Compare antibody signals in wild-type versus FASTKD2 knockdown/knockout cells.

• Overexpression controls: Detect overexpressed tagged FASTKD2 alongside endogenous protein.

• Molecular weight verification: Confirm observed molecular weight matches expected size (55-65 kDa) .

• Cross-reactivity testing: Test antibody against related FASTK family proteins to ensure specificity.

• Competing peptide blocking: Block antibody with immunogen peptide to confirm binding specificity.

These validation steps are essential before using FASTKD2 antibodies for critical experiments, especially when extending into new cell lines or tissues not previously tested.

What experimental approaches can be used to investigate FASTKD2's role in mitochondrial disease pathogenesis?

FASTKD2 mutations have been linked to mitochondrial encephalomyopathy . To study its role in disease pathogenesis:

• Patient-derived cells: Analyze FASTKD2 expression, mitochondrial transcript levels, and respiratory chain complex activities in cells from patients with FASTKD2 mutations.

• CRISPR/Cas9 disease modeling: Generate cell lines carrying patient-specific FASTKD2 mutations.

• Rescue experiments: Express wild-type FASTKD2 in patient cells to determine if defects are reversible.

• Mitochondrial function assays: Measure oxygen consumption, ATP production, and membrane potential in FASTKD2-deficient cells.

• Comprehensive mitochondrial transcriptome analysis: Use RNA-seq to identify dysregulated mitochondrial transcripts, similar to analyses performed for other FASTK family members .

• Apoptosis assays: Investigate whether FASTKD2 deficiency affects apoptotic response, given that other FAST domain proteins are involved in apoptotic pathways .

These approaches can help establish causality between FASTKD2 mutations and disease phenotypes, potentially revealing therapeutic targets.

What are the optimal dilutions and conditions for using HRP-conjugated FASTKD2 antibodies in Western blotting?

For optimal Western blot results with FASTKD2 antibodies:

• Dilution range: 1:500-1:2000 for primary antibody

• Sample preparation: Complete lysis of mitochondria is crucial; consider specialized mitochondrial isolation protocols followed by thorough lysis

• Gel percentage: 10-12% SDS-PAGE gels provide optimal resolution for the 55-65 kDa FASTKD2 protein

• Transfer conditions: Semi-dry or wet transfer at 100V for 60-90 minutes with methanol-containing transfer buffer

• Blocking: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Detection: For HRP-conjugated antibodies, ECL visualization systems are appropriate

• Exposure time: Start with short exposures (30 seconds) and increase as needed

The advantage of using HRP-conjugated primary antibodies is elimination of the secondary antibody incubation step, reducing protocol time and potentially lowering background.

How can researchers optimize immunofluorescence protocols for FASTKD2 localization studies?

For successful immunofluorescence studies of FASTKD2:

• Dilution range: 1:20-1:200

• Validated cell lines: COS7 and U2OS cells show positive staining

• Fixation: 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilization: 0.2% Triton X-100 for 10 minutes to ensure mitochondrial membrane permeabilization

• Blocking: 5% normal serum (goat or donkey) with 0.3% Triton X-100 for 1 hour

• Co-staining markers: Use established mitochondrial markers like mtSSB or MitoTracker dyes

• Mounting medium: Use anti-fade mounting medium containing DAPI for nuclear counterstaining

When studying FASTKD2's mitochondrial localization, confocal microscopy is recommended for optimal resolution of subcellular structures. Z-stack acquisition can help visualize the three-dimensional distribution of FASTKD2 within mitochondrial networks.

What controls should be included when using FASTKD2 antibodies in various experimental applications?

Proper controls are essential for reliable results with FASTKD2 antibodies:

Including these controls helps distinguish specific signals from background and validates experimental findings across different applications.

How can researchers quantify FASTKD2 expression levels across different experimental conditions?

Accurate quantification of FASTKD2 expression requires appropriate normalization and methodological considerations:

• Western blot quantification:

  • Normalize FASTKD2 signal to mitochondrial loading controls (e.g., VDAC, COX4)

  • Use infrared fluorescent secondary antibodies for wider linear range of detection

  • Include standard curves using recombinant FASTKD2 for absolute quantification

• RT-qPCR quantification:

  • Design primers specific to FASTKD2 mRNA

  • Normalize to stable reference genes (e.g., GAPDH was used in mouse studies)

  • Consider mitochondrial DNA content normalization for comparisons across tissues

• Immunofluorescence quantification:

  • Measure FASTKD2 fluorescence intensity within mitochondrial regions

  • Normalize to mitochondrial mass using mitochondrial markers

  • Use automated image analysis for unbiased quantification

• Proteomic approaches:

  • Targeted mass spectrometry with isotope-labeled peptide standards

  • Enrichment of mitochondrial fractions before analysis

These quantification methods can be applied to study FASTKD2 expression changes in response to mitochondrial stress, disease conditions, or genetic manipulations.

How do different FASTK family proteins interact in regulating mitochondrial RNA metabolism?

The FASTK family proteins (FASTK and FASTKD1-5) appear to have overlapping yet distinct functions in mitochondrial RNA processing . Research shows:

• FASTKD4 and FASTKD5 demonstrate overlapping functions in processing non-canonical junction sites of mitochondrial RNA, with combined knockout showing more severe phenotypes than single knockouts .

• FASTKD3 and FASTKD4 exert opposing effects on certain transcripts like ND3 mRNA, suggesting antagonistic regulatory mechanisms .

• FASTKD2 participates in a distinct complex involved in pseudouridylation , suggesting a specialized function compared to other family members.

Future research should focus on how these proteins coordinate their activities, whether they compete for binding to the same RNA targets, and how their expression levels are regulated in different tissues and physiological conditions. Co-immunoprecipitation experiments using FASTKD2 antibodies could help identify direct interactions with other family members.

What are the implications of FASTKD2 in broader mitochondrial disease contexts beyond the reported encephalomyopathy?

While FASTKD2 mutations have been specifically linked to mitochondrial encephalomyopathy , the protein's ubiquitous expression and role in fundamental mitochondrial RNA processing suggest potential implications in other mitochondrial disorders:

• FASTKD2's involvement in pseudouridylation of mitochondrial RNA suggests a role in post-transcriptional RNA modifications, which could affect translation efficiency of mitochondrial proteins.

• The observed cytochrome c oxidase (COX) deficiency in patients with FASTKD2 mutations indicates that FASTKD2 dysfunction may contribute to respiratory chain defects in other contexts.

• Given the FAST domain's involvement in apoptotic processes , FASTKD2 may link mitochondrial dysfunction to cell death pathways relevant in neurodegenerative disorders.

Future research should systematically analyze FASTKD2 expression and function in various mitochondrial disease models and patient samples beyond encephalomyopathy.

How might posttranslational modifications affect FASTKD2 function and antibody recognition?

Posttranslational modifications (PTMs) can significantly impact protein function and antibody binding. For FASTKD2:

• Potential phosphorylation sites exist throughout the protein, particularly in the FAST kinase domain, which could regulate its RNA binding activity.

• Mitochondrial proteins often undergo processing upon import, which could explain the discrepancy between predicted and observed molecular weights .

• Oxidative modifications may occur given the protein's mitochondrial localization and exposure to reactive oxygen species.

When using FASTKD2 antibodies, researchers should consider:

• Whether the antibody epitope contains potential PTM sites

• If treatment conditions might alter PTM status (e.g., phosphatase inhibitors)

• Whether different electrophoresis conditions might reveal multiple modified forms

Future phosphoproteomic and other PTM studies of FASTKD2 could provide insights into its regulation and potentially identify targetable modifications to modulate its function.