FASTKD2 Antibody

What is FASTKD2 and where is it expressed in tissues?

FASTKD2 (FAST kinase domains 2) is a mitochondrial protein that plays a crucial role in the assembly of the mitochondrial large ribosomal subunit. It functions as a component of a protein-RNA module that controls 16S mitochondrial ribosomal RNA (16S mt-rRNA) abundance and is required for intra-mitochondrial translation . Expression analysis reveals that FASTKD2 is ubiquitously expressed throughout the body, with notably higher expression levels observed in tissues with high-energy demands, including the brain, heart, and skeletal muscle . This expression pattern correlates with its fundamental role in ensuring mitochondria maintain energetic efficiency through effective RNA management .

What applications are validated for FASTKD2 antibodies?

FASTKD2 antibodies have been validated for multiple experimental applications across various sample types:

The antibody has been tested with positive results in multiple cell lines including HeLa, HepG2, and K-562 cells for Western blot applications, demonstrating its versatility across human cell types . When designing experiments, researchers should always perform antibody titration in each specific testing system to obtain optimal results, as the ideal concentration can be sample-dependent .

How should researchers optimize Western blot protocols for FASTKD2 detection?

For optimal Western blot results with FASTKD2 antibodies, researchers should consider the following methodological approach:

• Sample preparation: Prepare lysates from tissues with known FASTKD2 expression (heart, brain, or cell lines like HeLa or HepG2) using a lysis buffer containing protease inhibitors to prevent degradation .

• Protein loading: Load 20-40 μg of total protein per lane for cell lines, and up to 60 μg for tissue samples.

• Gel selection: Use 8-10% SDS-PAGE gels for optimal separation, considering that the predicted molecular weight of FASTKD2 is 81 kDa, although the observed molecular weight in experimental conditions often appears between 55-65 kDa .

• Transfer conditions: Transfer proteins to PVDF or nitrocellulose membranes using standard protocols (100V for 1 hour or 30V overnight).

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

• Primary antibody incubation: Dilute FASTKD2 antibody between 1:500-1:2000 in blocking buffer and incubate overnight at 4°C with gentle agitation .

• Detection: For polyclonal rabbit FASTKD2 antibodies, use anti-rabbit IgG secondary antibodies (1:50000 dilution has been validated) .

The predicted band size for FASTKD2 is 81 kDa, but researchers should be aware that the observed molecular weight may vary between 55-65 kDa in many samples . This discrepancy could be due to post-translational modifications or processing of the mitochondrial targeting sequence.

What are the best practices for immunofluorescence studies using FASTKD2 antibodies?

For immunofluorescence detection of FASTKD2, researchers should implement the following procedure:

• Cell culture: Grow cells on coverslips until 70-80% confluent. COS7 and U2OS cells have been validated for FASTKD2 immunofluorescence studies .

• Fixation: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature.

• Permeabilization: Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes to allow antibody access to mitochondrial proteins.

• Blocking: Block with 3-5% BSA in PBS for 1 hour at room temperature.

• Primary antibody: Dilute FASTKD2 antibody at 1:20-1:200 in blocking solution and incubate overnight at 4°C .

• Mitochondrial co-staining: For colocalization studies, co-stain with established mitochondrial markers such as anti-mtSSB (mitochondrial single-strand-DNA binding protein) antibody .

• Secondary antibody: Incubate with fluorophore-conjugated secondary antibodies at appropriate dilutions for 1 hour at room temperature.

• Imaging: Acquire images using confocal microscopy for optimal resolution of mitochondrial structures.

FASTKD2 has been experimentally verified to localize to mitochondria through co-localization with known mitochondrial markers . When FASTKD2 was tagged with hemagglutinin (HA) and expressed in COS7 and HeLa cells, the immunofluorescence pattern completely overlapped with mitochondrial markers, confirming its mitochondrial localization .

What is the relationship between FASTKD2 and mitochondrial disease pathology?

FASTKD2 mutations have been implicated in mitochondrial encephalomyopathy characterized by developmental delay, hemiplegia, convulsions, asymmetrical brain atrophy, and low cytochrome c oxidase (COX) activity in skeletal muscle . The pathophysiological mechanism involves:

• Protein truncation: A homozygous nonsense mutation (c.1246C→T) causing R416X premature termination codon has been identified in patients with mitochondrial encephalomyopathy . This mutation results in a truncated protein missing 278 of the 694 C-terminal amino acid residues, including two predicted transmembrane domains (positions 403-421 and 482-500) and the FAST kinase domain .

• Mitochondrial translation: FASTKD2 is part of a functional protein-RNA module controlling 16S mt-rRNA abundance and is required for intra-mitochondrial translation . Loss of function impacts mitochondrial protein synthesis.

• Apoptotic regulation: Experimental evidence suggests FASTKD2 plays a specific role in regulating mitochondrial apoptosis. Fibroblasts from patients with FASTKD2 mutations showed significantly lower percentages of fragmented nuclei compared to control cells when treated with staurosporine, an apoptosis inducer . Conversely, overexpression of recombinant FASTKD2-HA in HeLa or COS7 cells led to cell body shrinkage, nuclear fragmentation, and eventual apoptotic death .

When studying FASTKD2 in disease models, researchers should consider using antibodies that recognize epitopes outside the commonly mutated regions to ensure detection of truncated proteins for comparative studies.

How can the discrepancy between predicted and observed molecular weights of FASTKD2 be investigated?

The predicted molecular weight of FASTKD2 is 81 kDa, but the observed molecular weight in experimental conditions often ranges between 55-65 kDa . Researchers investigating this discrepancy should consider the following methodological approaches:

• Mitochondrial import processing: FASTKD2 contains a mitochondrial targeting sequence that is cleaved upon import into mitochondria. Experimental evidence has shown that when exposed to energized mitochondria, radiolabeled FASTKD2 produces two bands: a larger band corresponding to the full-length protein and a shorter one corresponding to the mature protein after cleavage of the N-terminal mitochondrial targeting sequence .

• Protease protection assay: The potential cleavage site of the 79 kDa FASTKD2 precursor protein by the mitochondrial matrix protease (MMP) appears to be between M52 and Q53, predicting a mature imported protein of approximately 73 kDa (642 amino acid residues) . This can be verified by treating isolated mitochondria containing FASTKD2 with Proteinase K and observing which fragments are protected.

• Alternative translation start site analysis: Evidence suggests that in humans and other species, the "true" FASTKD2 protein may start from M17 rather than from M1 . When mitochondrial-targeting prediction software is used, the sequence starting at M17 scores much higher for mitochondrial targeting (Mitoprot: 91% versus 72%; TargetP: 76% versus 43%) .

• Western blot with multiple antibodies: Researchers should use antibodies targeting different epitopes of FASTKD2 to determine if specific regions are consistently detected across different samples and experimental conditions.

• Mass spectrometry analysis: To definitively identify the mature form of FASTKD2, immunoprecipitate the protein and analyze by mass spectrometry to determine its exact molecular composition and post-translational modifications.

Understanding these molecular weight variations is crucial for correctly interpreting experimental results and ensuring consistent identification of FASTKD2 across different studies.

What controls should be included when validating FASTKD2 antibody specificity?

For rigorous validation of FASTKD2 antibody specificity, researchers should incorporate these essential controls:

• Genetic knockout/knockdown validation: CRISPR/Cas9-mediated gene editing can generate FASTKD2 knockout cell lines, such as those derived from 143B cells as described for FASTKD5 studies . The knockout should be confirmed by genomic sequencing and used as a negative control to verify antibody specificity. Similar approaches using siRNA or shRNA knockdown provide alternative validation methods.

• Overexpression controls: Complementary to knockout models, overexpression of tagged FASTKD2 (e.g., with FLAG or HA tags) in wild-type or knockout backgrounds can verify antibody recognition of the target protein .

• Peptide competition assay: Pre-incubating the antibody with the immunizing peptide before applying to samples should abolish specific signals if the antibody is truly specific.

• Cross-reactivity assessment: Test the antibody against other FASTK family members (FASTK, FASTKD1, FASTKD3-5) to ensure it doesn't cross-react with structurally similar proteins.

• Multiple application validation: Verify specificity across different applications (WB, IF, IHC) as non-specific binding can manifest differently depending on the technique.

• Species validation: For studies involving multiple species, confirm specificity in each species of interest, especially if studying evolutionary conserved functions.

• Tissue-specific expression correlation: Compare antibody detection patterns with known tissue expression profiles of FASTKD2, which should show higher levels in brain, heart, and skeletal muscle .

What are the optimal conditions for studying FASTKD2's role in mitochondrial apoptosis?

To investigate FASTKD2's role in mitochondrial apoptosis, researchers should consider these methodological approaches:

• Apoptosis induction: Use staurosporine (1 mM for 3 hours), a potent protein kinase C inhibitor that induces apoptosis through a mitochondria-mediated pathway and causes oxidative stress through mitochondrion-generated ROS .

• FASTKD2 manipulation: Compare responses between:

  • Wild-type cells with normal FASTKD2 expression

  • FASTKD2 knockout/knockdown cells

  • Cells with FASTKD2 mutations (e.g., R416X)

  • Cells overexpressing wild-type or mutant FASTKD2

• Apoptosis assessment: Evaluate apoptosis using multiple complementary methods:

  • Nuclear fragmentation quantification

  • Caspase-3/7 activity assays

  • Annexin V/PI staining and flow cytometry

  • Cytochrome c release from mitochondria

  • Mitochondrial membrane potential measurements

• Mitochondrial function assessment: Monitor respiratory chain complex activities alongside apoptotic markers to distinguish FASTKD2-specific effects from general mitochondrial dysfunction .

• Temporal analysis: Track the sequence of events in a time-course experiment after apoptosis induction to determine when and how FASTKD2 influences the apoptotic cascade.

• Protein-protein interaction studies: Investigate FASTKD2 interactions with known apoptotic regulators using co-immunoprecipitation or proximity ligation assays.

When designing these experiments, remember that overexpression of recombinant FASTKD2-HA in cell models has been shown to induce cell shrinkage, nuclear fragmentation, and eventual apoptotic death , suggesting careful calibration of expression levels is needed for meaningful results.

What methodologies are appropriate for studying FASTKD2's interactions with the mitochondrial ribosome?

To investigate FASTKD2's interactions with the mitochondrial ribosome, researchers should implement these specialized approaches:

• Co-immunoprecipitation (Co-IP): Use anti-FASTKD2 antibodies to pull down protein complexes, followed by detection of mitochondrial ribosomal proteins. Recommended conditions include:

  • Gentle lysis buffers containing 1% digitonin or 0.5% NP-40 to preserve protein-protein interactions

  • DNase and RNase treatments to distinguish RNA-dependent interactions

  • Crosslinking steps for transient interactions

• RNA immunoprecipitation (RIP): FASTKD2 antibodies can be used for RIP assays to identify associated RNAs, particularly 16S mitochondrial ribosomal RNA . This technique has been validated in previous studies of FASTK family proteins .

• Sucrose gradient fractionation: Separate mitochondrial ribosomal subunits and intact ribosomes on sucrose gradients, followed by immunoblotting for FASTKD2 to determine its association with specific ribosomal components.

• Proximity labeling: Use techniques like BioID or APEX2 fused to FASTKD2 to identify proximal proteins in the native mitochondrial environment.

• Cryo-electron microscopy: For structural studies of FASTKD2 within the context of the mitochondrial ribosome.

• Functional assays: Measure mitochondrial translation efficiency using pulse-labeling with 35S-methionine in FASTKD2 wildtype versus knockout or mutant cells to assess functional consequences of FASTKD2's interactions with the mitochondrial ribosome.

FASTKD2 plays an important role in the assembly of the mitochondrial large ribosomal subunit and, as part of a functional protein-RNA module (including RCC1L, NGRN, RPUSD3, RPUSD4, TRUB2, and 16S mt-rRNA), controls 16S mt-rRNA abundance required for intra-mitochondrial translation . Therefore, these methodological approaches will help elucidate the molecular mechanisms underpinning this critical function.