FASTK Antibody

What is FASTK and why is it important in research?

FASTK (Fas activated serine/threonine kinase) is a 549 amino acid protein with a mass of 61.1 kDa that localizes primarily to mitochondria in humans. It belongs to the FAST protein kinase family and plays significant roles in apoptotic pathways and protein phosphorylation. Its importance stems from its wide expression pattern across vital tissues including heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. The protein undergoes post-translational modifications, particularly phosphorylation, making it a valuable research target for understanding cellular signaling and disease mechanisms .

What are the most effective applications for FASTK antibodies in basic research?

FASTK antibodies have demonstrated highest utility in three primary applications: Western Blot (WB), Enzyme-Linked Immunosorbent Assay (ELISA), and Immunohistochemistry (IHC). Western Blot remains the most widely used application, allowing researchers to detect and quantify FASTK protein in tissue lysates, while immunohistochemistry provides crucial spatial information about protein localization within tissues. When designing experiments, researchers should consider these validated applications for optimal results rather than attempting less established methods .

How should researchers design experiments to investigate FASTK's role in immune cell infiltration?

When investigating FASTK's relationship with immune infiltration, researchers should employ a multi-omics approach. Based on recent studies of FASTK in kidney renal clear cell carcinoma (KIRC), experimental designs should incorporate:

• RNA-seq data analysis using fragments per kilobase per million fragments mapped (FPKM) or transcripts per million (TPM) methods

• Single-cell analysis platforms such as CancerSEA for functional status assessment

• Correlation analysis with established immune cell markers

• Validation across multiple databases (TCGA, GEO datasets, etc.)

The experimental workflow should follow a pattern similar to that used in recent KIRC studies, moving from expression analysis to correlation with immune infiltration markers .

What methodological considerations are critical when using FASTK antibodies for studying different members of the FASTK family?

The FASTK family includes multiple members (FASTK, FASTKD1, FASTKD2, FASTKD3, TBRG4, FASTKD5) with distinct but potentially overlapping functions. When designing antibody-based experiments:

• Validate antibody specificity against each family member using knockout/knockdown controls

• Consider epitope locations to ensure discrimination between family members

• Employ antibodies targeting different regions (N-terminal vs. C-terminal) for confirmation

• Use orthogonal detection methods (mRNA quantification) to support protein-level findings

How should researchers approach contradictions in FASTK expression data across different cancer types?

When encountering contradictory FASTK expression data across cancer types:

• Compare normalization methods used in different studies (FPKM vs. TPM)

• Examine cohort demographics and tumor characteristics

• Consider tumor heterogeneity and the presence of distinct molecular subtypes

• Analyze correlation with patient outcomes rather than absolute expression levels

• Perform immune cell deconvolution to account for infiltrating immune component contributions

Recent studies in KIRC demonstrate the importance of correlating FASTK expression with tumor immune microenvironment features rather than focusing solely on expression levels .

What are the optimal sample preparation protocols for FASTK detection in different applications?

For optimal FASTK detection:

Western Blot:

• Use RIPA buffer with protease and phosphatase inhibitors

• Include reducing agents (β-mercaptoethanol) in sample buffer

• Heat samples at 95°C for 5 minutes before loading

• Transfer using semi-dry methods at 15V for 1 hour for optimal protein transfer

Immunohistochemistry:

• Use 10% neutral buffered formalin fixation (24 hours)

• Perform antigen retrieval using citrate buffer (pH 6.0)

• Block with 5% normal serum corresponding to secondary antibody species

• Optimize primary antibody dilution (typically 1:100-1:500)

ELISA:

• Extract protein using mild detergent buffers

• Dilute samples to fall within standard curve range

• Follow specific manufacturer recommendations for coating buffers

These protocols should be optimized based on tissue type and experimental conditions .

How can researchers effectively validate FASTK antibody specificity for their experimental system?

A comprehensive FASTK antibody validation approach should include:

• Positive and negative control tissues (based on known expression patterns)

• FASTK knockdown/knockout controls when possible

• Peptide competition assays to confirm epitope specificity

• Detection of expected molecular weight (61.1 kDa) in Western blot

• Correlation of protein data with mRNA expression

• Comparison of results using antibodies against different epitopes

• Testing in multiple applications (WB, IHC, etc.) to confirm consistent results

Proper validation is particularly important given the multiple FASTK family members and potential splice variants .

What are the key considerations for investigating FASTK's interaction with immune cells in tumor microenvironments?

When investigating FASTK's role in tumor immune microenvironments:

• Use single-cell RNA sequencing to distinguish cell type-specific expression

• Apply multiplex immunofluorescence to simultaneously visualize FASTK and immune markers

• Employ immune deconvolution algorithms (CIBERSORT, xCell, etc.) for bulk RNA-seq data

• Consider spatial transcriptomics to map FASTK expression relative to immune niches

• Correlate FASTK expression with specific immune cell markers using methods like:

The above markers have been validated in KIRC studies and should be considered for comprehensive immune profiling .

How should researchers interpret contradictory results between FASTK protein detection and gene expression data?

When facing discrepancies between protein and mRNA levels:

• Consider post-transcriptional regulation mechanisms, particularly miRNAs targeting FASTK

• Evaluate protein stability and half-life factors

• Assess technical limitations (antibody sensitivity vs. RNA-seq depth)

• Examine potential alternative splicing affecting epitope availability

• Consider cell type heterogeneity in bulk samples

A combined approach analyzing both protein (Western blot/IHC) and mRNA (qPCR/RNA-seq) is recommended to resolve contradictions. Researchers should also calculate correlation coefficients between protein and mRNA measurements across samples to quantify the extent of discordance .

What statistical approaches are most appropriate for analyzing the correlation between FASTK expression and immune infiltration?

For robust statistical analysis of FASTK and immune infiltration correlations:

• Use Spearman correlation for non-parametric assessment of monotonic relationships

• Apply Wilcoxon signed-rank sum test for comparing high vs. low FASTK expression groups

• Employ Single-sample Gene Set Enrichment Analysis (ssGSEA) to quantify immune infiltration

• Consider multivariate regression to account for confounding clinical variables

• Validate findings through multiple computational tools (TIMER, TISIDB, etc.)

• Set significance threshold at p < 0.05 with appropriate multiple testing corrections

• Visualize correlations using heatmaps and scatter plots with regression lines

These statistical approaches have been successfully applied in KIRC studies and provide a framework for other cancer types .

How can researchers differentiate between direct and indirect effects of FASTK on immune cell function?

To distinguish direct from indirect FASTK effects on immune function:

• Perform conditional knockout experiments targeting FASTK in specific cell populations

• Use co-culture systems with FASTK-modulated cells and immune components

• Analyze protein-protein interaction networks using STRING database

• Examine gene-gene interaction networks with GeneMANIA

• Apply causal inference statistical methods (e.g., mediation analysis)

• Consider temporal dynamics through time-course experiments

• Evaluate signaling pathway activation downstream of FASTK

Understanding these relationships requires integrating both computational predictions and experimental validation to establish causality rather than mere correlation .

What emerging technologies show promise for advancing FASTK research beyond traditional antibody-based methods?

Cutting-edge technologies that complement antibody-based FASTK research include:

• CRISPR-Cas9 gene editing for creating precise FASTK mutants

• Proximity labeling methods (BioID, APEX) to identify novel FASTK interactors

• Mass spectrometry-based phosphoproteomics to characterize FASTK substrates

• Single-cell multi-omics to correlate FASTK expression with cell states

• Spatial transcriptomics to map FASTK expression in tissue microenvironments

• Cryo-EM for structural studies of FASTK complexes

• Live-cell imaging with FASTK-fluorescent protein fusions

These technologies address limitations of antibody-based approaches and provide complementary data for comprehensive FASTK characterization .

How should researchers approach the study of FASTK family members across different cancer types?

For comparative oncology studies of FASTK family members:

This systematic approach allows researchers to identify both common and unique roles of FASTK family members across cancer types .

What methodological considerations are important when studying FASTK's role in the mitochondria?

For mitochondrial FASTK research:

• Use appropriate subcellular fractionation techniques to isolate pure mitochondria

• Employ mitochondria-specific markers (TOMM20, COX4) as controls in co-localization studies

• Consider mitochondrial dynamics (fusion/fission) when interpreting FASTK localization patterns

• Assess mitochondrial function parameters (membrane potential, respiration, ROS production)

• Analyze mitochondrial morphology changes upon FASTK modulation

• Study mtDNA-encoded gene expression as potential FASTK targets

• Consider cell type-specific variations in mitochondrial content and function

Given FASTK's mitochondrial localization, careful attention to mitochondrial isolation quality and appropriate controls is essential for accurate interpretation of results .