GK5 Antibody

What is GK5 and what specific epitopes are targeted by commercial antibodies?

GK5 (Glycerol Kinase 5) is a skin-specific kinase that plays a key role in glycerol metabolism, catalyzing the phosphorylation of glycerol to produce sn-glycerol 3-phosphate. This enzyme is involved in skin-specific regulation of sterol regulatory element-binding protein (SREBP) processing and lipid biosynthesis . GK5 belongs to the FGGY kinase family and exists as three isoforms produced by alternative splicing with molecular weights of 59 kDa, 34 kDa, and 28 kDa .

Commercial antibodies target various epitopes of GK5:

• N-terminal region (aa 1-300)

• Middle region (aa 250-400)

• C-terminal region

The immunogen sequences for specific antibodies include:

• HPA057998: "QQSAMFGECCFQTGDVKLTMGTGTFLDINTGNSLQQTTGGFYPLIGWKIGQEVVCLAESNAGDTGTAIKWAQQLDLFTDAAET"

• HPA042606: "NCCFGTIDTWLLYKLTKGSVYATDFSNASTTGLFDPYKMCWSGMITSLISIPLSLLPPVRDTSHNFGSVDEEIFGVPIPIVAL"

What validation methods should be used to confirm GK5 antibody specificity?

A multi-tiered validation approach is recommended:

Primary validation techniques:

• Western blot analysis using positive control tissues (e.g., skin, sebaceous glands) and negative control tissues (e.g., liver)

• Protein array testing against 384 different antigens including the antibody target

• Immunohistochemistry comparison across multiple tissues to confirm expected expression pattern

Enhanced validation techniques:

• siRNA knockdown: Evaluate decrease in antibody-based staining intensity upon target protein downregulation

• GFP validation: Assess signal overlap between antibody staining and GFP-tagged GK5 protein

• Independent antibody validation: Compare staining patterns of two or more antibodies directed toward different epitopes of GK5

How should researchers design experiments to study GK5's role in SREBP processing and lipid biosynthesis?

To effectively study GK5's regulatory role in SREBP processing and lipid homeostasis, a comprehensive experimental approach is required:

Recommended experimental workflow:

• Establish baseline GK5 expression using validated antibodies in primary sebocytes or skin tissue

• Use GK5 antibodies to co-immunoprecipitate protein complexes containing SREBPs to confirm protein-protein interactions

• Implement siRNA knockdown of GK5 to examine effects on:

  • SREBP nuclear translocation (using fractionation and immunoblotting)

  • Lipid synthesis pathways (using metabolic labeling techniques)

  • Cholesterol, triglyceride, and ceramide accumulation

• Compare results with simvastatin treatment, which partially rescues phenotypes in GK5-deficient models

Critical controls:

• Include tissue from GK5-knockout models where available

• Validate antibody specificity in each experimental system

• Include both kinase-inactive GK5 mutants and complete knockout conditions to distinguish between enzymatic and scaffolding functions

What approaches should be used to distinguish between GK5 isoforms in experimental systems?

GK5 exists in three isoforms (59 kDa, 34 kDa, and 28 kDa) , requiring careful experimental design to distinguish between them:

Isoform-specific detection strategy:

• Use antibodies targeting different regions of GK5 to detect specific isoforms

• Implement RT-PCR with isoform-specific primers to correlate protein detection with transcript expression

• Perform subcellular fractionation to identify potential compartment-specific localization of different isoforms

• Use mass spectrometry to confirm the identity of immunoprecipitated isoforms

Methodological considerations:

• When using Western blot, include positive controls expressing each isoform separately

• Consider the observed versus expected molecular weights (the actual band is not always consistent with expectations)

• Use gradient gels (4-15%) to better resolve the different isoforms

How can computational approaches enhance GK5 antibody design and specificity?

Recent advances in computational methods offer powerful tools for designing antibodies with enhanced specificity for GK5:

Computational design workflow:

• Begin with phage display experiments selecting antibodies against multiple ligands to create training data

• Implement a biophysics-informed model that associates distinct binding modes with each potential ligand

• Use the model to predict antibody variants with customized specificity profiles:

  • High affinity for GK5 with minimal cross-reactivity to other glycerol kinase family members

  • Specific binding to selected GK5 domains or isoforms

• Generate and experimentally validate the computationally designed antibody sequences

Advantages demonstrated in research:

• The computational approach can disentangle binding modes even for chemically similar ligands

• Models can predict outcomes for new ligand combinations beyond the training set

• This approach has been shown to reduce the number of required antigen variants by up to 35%

• The method speeds up the learning process by approximately 28 steps compared to random selection baselines

What are the optimal protocols for using GK5 antibodies in immunohistochemistry and immunofluorescence?

Immunohistochemistry (IHC) Protocol:

• Tissue preparation: Use formalin-fixed, paraffin-embedded (FFPE) sections (4-6 μm thickness)

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Antibody dilution:

  • HPA042606: 1:20-1:50 for IHC

  • E-AB-18674: 1:30-1:150 for IHC

• Detection system: Use HRP-conjugated secondary antibodies with DAB substrate

• Validated positive controls: Human skin, sebaceous glands, or esophagus cancer tissue

Immunofluorescence (IF) Protocol:

• Cell preparation: PFA-fixed, Triton X-100 permeabilized cells

• Antibody dilution:

  • HPA057998: 0.25-2 μg/mL for IF

  • ab220869: 4 μg/mL for IF

• Detection: Use appropriate fluorophore-conjugated secondary antibodies

• Validated cell lines: SiHa cells have been confirmed to express detectable levels of GK5

What methodologies are recommended for studying GK5's role in pathological conditions?

For investigating GK5's involvement in disease states such as hair loss, metabolic disorders, or skin conditions:

Experimental approach:

• Patient sample analysis:

  • Compare GK5 expression levels between healthy and pathological tissues using validated antibodies

  • Correlate GK5 expression with lipid profiles and SREBP activation markers

  • Analyze GK5 genetic variants in patient populations

• Functional studies:

  • Use GK5 antibodies to monitor protein levels after treatment with metabolic regulators

  • Implement GK5 knockdown or overexpression in relevant cell models

  • Assess effects on:

    • Cholesterol biosynthesis pathway

    • Triglyceride synthesis

    • Ceramide production

    • Hair follicle development in 3D skin models

• Therapeutic intervention testing:

  • Monitor GK5 expression and activity during treatment with lipid metabolism modulators

  • Use GK5 antibodies to track protein-protein interactions in response to treatment

  • Evaluate combinatorial approaches targeting both GK5 and SREBP pathways

Notable research finding: GK5-deficient mice display alopecia (hair loss) due to impaired hair growth and maintenance, which can be partially rescued by treatment with the HMG-CoA reductase inhibitor simvastatin .

How should researchers address cross-reactivity concerns when using GK5 antibodies?

Cross-reactivity is a critical concern when studying GK5, particularly due to its homology with other glycerol kinase family members:

Cross-reactivity assessment workflow:

• In silico analysis:

  • Check sequence homology between GK5 and other glycerol kinases

  • Verify immunogen sequence uniqueness using BLAST analysis

  • Review the Human Protein Atlas maximum percent sequence identity data for the antibody target region

• Experimental validation:

  • Perform Western blot analysis in tissues expressing multiple glycerol kinase family members

  • Include positive and negative control lysates

  • Test antibody against recombinant GK1-5 proteins

  • Consider using protein array validation containing 384 different antigens

• Controls to implement:

  • Include GK5 knockout or knockdown samples

  • Compare results using at least two antibodies targeting different epitopes

  • Consider using GK5-GFP fusion proteins as positive controls

Key considerations from research:

• The immunogen sequence should ideally have <60% identity to other proteins for designing a single-target antibody

• Regions with lowest possible identity to other proteins should be selected for antibody generation

• Consider antibody validation scores: Enhanced, Supported, Approved, or Uncertain

How can active learning approaches improve experimental efficiency in GK5 antibody research?

Active learning methodologies can significantly enhance the efficiency of antibody research by optimizing experimental design:

Active learning implementation strategy:

• Start with a small labeled subset of antibody-antigen binding data

• Use computational models to predict binding for unlabeled pairs

• Select the most informative experiments to perform next based on uncertainty or expected information gain

• Iterate the process, continuously improving the model with new experimental data

Demonstrated benefits:

• Reduction in required antigen mutant variants by up to 35%

• Acceleration of the learning process by 28 steps compared to random selection

• Improved out-of-distribution prediction performance for antibody-antigen binding

• Enhanced ability to design antibodies with customized specificity profiles

Implementation considerations:

• Three specific active learning algorithms have been shown to significantly outperform random selection in antibody-antigen binding prediction

• The approach is particularly valuable for library-on-library screening approaches with many-to-many relationships

• This methodology is especially useful when test antibodies and antigens are not represented in training data (out-of-distribution prediction)

What experimental approaches should be used to investigate GK5's interaction with SREBPs and regulation of lipid biosynthesis?

To elucidate the molecular mechanisms of GK5's regulation of SREBP processing:

Comprehensive investigation protocol:

• Protein-protein interaction analysis:

  • Co-immunoprecipitation using GK5 antibodies followed by SREBP detection

  • Proximity ligation assays to visualize interactions in situ

  • FRET/BRET analysis using tagged proteins to assess direct interactions

• Functional domain mapping:

  • Use GK5 antibodies against different epitopes to identify interaction interfaces

  • Implement domain deletion/mutation constructs to identify critical regions

  • Assess kinase-dependent vs. kinase-independent functions using catalytically inactive mutants

• Regulatory pathway analysis:

  • Monitor SREBP cleavage and nuclear translocation in response to GK5 manipulation

  • Assess effects on downstream lipid synthesis genes using RT-qPCR

  • Quantify cholesterol, triglyceride, and ceramide levels using mass spectrometry

Key research findings:

• GK5 forms a complex with SREBPs through their C-terminal regulatory domains

• This interaction inhibits SREBP processing and activation

• In GK5-deficient mice, transcriptionally active SREBPs accumulate in skin (but not liver), leading to elevated lipid synthesis

• Both kinase activity and protein-protein interactions appear important, as kinase-inactive GK5 mutants also show defective hair growth

How can researchers evaluate potential off-target effects when using GK5 antibodies in complex experimental systems?

Ensuring antibody specificity in complex systems requires rigorous validation:

Comprehensive validation workflow:

• Multi-platform approach:

  • Compare results across different detection methods (WB, IHC, IF, IP)

  • Validate findings using orthogonal approaches (RNA expression, activity assays)

  • Implement at least two independent antibodies targeting different GK5 epitopes

• Controls to implement:

  • Genetic knockout/knockdown of GK5 in the experimental system

  • Pre-absorption controls using the immunizing peptide

  • Include tissues/cells known to be negative for GK5 expression

• Advanced validation techniques:

  • Mass spectrometry analysis of immunoprecipitated proteins

  • ChIP-MS to identify antibody interactions in chromatin contexts

  • Protein array screening against 384 different antigens

Notable validation parameters from research:

• Standard validation should confirm concordance with UniProtKB/Swiss-Prot database information

• Enhanced validation includes siRNA knockdown, GFP-tagged cell lines, or independent antibody comparisons

• Western blot bands should be evaluated against the predicted size (59 kDa for full-length GK5)

• Consider that modified forms of the protein may result in bands that differ from the expected size