GMFB His Human

What are the primary functions of GMFB in neural tissues?

GMFB serves multiple crucial functions in neural tissues:

• Neural differentiation: GMFB is involved in the differentiation of both glial cells and neurons .

• Axon regeneration: It stimulates neural regeneration processes, making it of interest in neurological injury research .

• Anti-proliferative effects: GMFB reversibly inhibits proliferation of neuronal and non-neuronal neoplastic cells by arresting the cell cycle in the G0/G1 phase .

• Signal transduction: It functions as an intracellular regulator of signal pathways, particularly in astrocytes .

• Interaction mechanism: GMFB is expressed in the cytosol and on the cell surface of astrocytes and interacts with target receptors in a juxtacrine manner .

Methodologically, when studying these functions, researchers should carefully consider the protein's context-dependent activities and ensure appropriate controls when using recombinant His-tagged versions versus native protein.

How is GMFB distributed across different tissues and what methods can detect its presence?

GMFB shows a distinctive tissue distribution pattern that researchers can analyze using enzyme immunoassays (EIAs) developed specifically for GMFB and GMFG detection .

Tissue Distribution of GMFB in Rat and Human:

For detection and quantification, researchers have developed two-site enzyme immunoassays using specific antibodies raised in rabbits . These assay systems are highly sensitive and can detect GMFB in both human and rat samples, allowing for accurate measurement in various tissues and serum .

When designing experiments to study GMFB tissue distribution, researchers should:

• Consider cross-reactivity with GMFG

• Include appropriate controls

• Use sufficiently sensitive detection methods (the EIA systems mentioned have demonstrated adequate sensitivity)

• Account for potential age-related variations in serum levels

What storage and handling conditions are recommended for recombinant GMFB His protein?

Proper storage and handling of recombinant GMFB His protein is essential for maintaining its stability and activity. Based on manufacturer recommendations:

Storage Conditions:

• Store desiccated at -20°C for long-term storage

• For frequent use within 2-4 weeks, store at 4°C

• Avoid repeated freeze-thaw cycles as these can degrade the protein

Buffer and Reconstitution:

• Typical buffer composition: 20mM Tris-HCL pH 8.0, 1mM DTT, 0.1M NaCl, and 10% Glycerol

• Follow specific reconstitution instructions provided by the manufacturer

• Once reconstituted, aliquot to minimize freeze-thaw cycles

Quality Considerations:

• Confirm endotoxin levels before use in cell culture applications

• Verify protein purity (typically >95% by SDS-PAGE)

• Consider testing bioactivity before use in functional assays

When planning experiments, researchers should account for the impact of storage conditions on protein activity and document the storage history of samples used.

How does SUMOylation affect GMFB stability and function?

SUMOylation represents an important post-translational modification that impacts GMFB stability and function, particularly under stress conditions. Research has identified that the expression of both GMFB and SUMO1 is enhanced during the early stages of hypoxia/high glucose or oxidative stress .

Methodological approach to study SUMOylation of GMFB:

• Identification of SUMOylation sites:

  • Use site-directed mutagenesis to modify potential SUMOylation sites

  • Employ mass spectrometry to confirm modification sites

  • Apply in silico prediction tools in combination with experimental validation

• Functional impact assessment:

  • Compare wild-type and SUMOylation-deficient GMFB mutants in functional assays

  • Measure protein half-life to determine stability changes

  • Analyze subcellular localization patterns before and after stress induction

• Stress-response studies:

  • Create controlled hypoxia and high glucose conditions in cell culture

  • Generate oxidative stress using H₂O₂ or other oxidizing agents

  • Monitor GMFB and SUMO1 expression levels using quantitative techniques

  • Track temporal changes in SUMOylation patterns during stress response

When conducting SUMOylation studies, researchers should control for other post-translational modifications that might occur simultaneously, such as phosphorylation, which is also known to affect GMFB .

What methodologies are most effective for studying GMFB's role in axon regeneration?

GMFB's capacity to stimulate axon regeneration makes it a promising target for neurological injury research. Effective methodological approaches include:

In vitro methods:

• Primary neuronal cultures:

  • Establish cultures of primary neurons from appropriate brain regions

  • Apply purified GMFB His protein at varying concentrations (typically 10-100 ng/ml)

  • Quantify axon outgrowth using immunofluorescence and morphometric analysis

  • Compare His-tagged versus untagged GMFB to account for tag interference

• Organotypic slice cultures:

  • Create lesions in brain or spinal cord slices

  • Treat with recombinant GMFB His protein

  • Track regeneration using time-lapse microscopy and axon-specific markers

In vivo methods:

• Injury models:

  • Implement standardized spinal cord or peripheral nerve injury procedures

  • Deliver GMFB using appropriate vehicles (hydrogels, nanoparticles, etc.)

  • Assess functional recovery using behavioral testing

  • Perform histological analysis to quantify axon regeneration

• Genetic approaches:

  • Use GMFB knockout or conditional knockout models

  • Implement CRISPR-Cas9 for tissue-specific editing

  • Employ viral vectors for localized overexpression or knockdown

Assessment techniques:

• Anterograde and retrograde tracing to visualize regenerating axons

• Electrophysiological recordings to assess functional connectivity

• Serial sectioning and 3D reconstruction to quantify regeneration

• Molecular markers of regeneration (GAP-43, etc.)

When designing these experiments, researchers should consider the context-dependent activities of GMFB and potential interactions with other factors involved in axon regeneration.

What experimental approaches are recommended for investigating GMFB's tumor inhibition properties?

GMFB has demonstrated the ability to inhibit tumor cell proliferation by arresting the cell cycle in the G0/G1 phase . Investigating this property requires a multifaceted approach:

Cell proliferation studies:

• Growth curve analysis:

  • Treat various tumor cell lines with recombinant GMFB His protein

  • Monitor cell numbers over time using automated cell counters or cell viability assays

  • Establish dose-response relationships (typically testing 1-100 μg/ml)

  • Determine reversibility by removing GMFB and monitoring recovery

• Cell cycle analysis:

  • Use flow cytometry with propidium iodide or other DNA stains

  • Analyze the percentage of cells in G0/G1, S, and G2/M phases

  • Implement BrdU incorporation assays to confirm S-phase inhibition

  • Apply synchronization techniques to highlight cell cycle effects

Molecular mechanism investigation:

• Transcriptomic analysis:

  • Perform RNA-seq on GMFB-treated versus untreated tumor cells

  • Focus on cell cycle regulatory genes and pathways

  • Validate key findings with qRT-PCR

• Proteomic studies:

  • Use mass spectrometry to identify GMFB-interacting proteins

  • Examine phosphorylation status of key cell cycle regulators

  • Implement co-immunoprecipitation to confirm specific interactions

• Signal transduction analysis:

  • Monitor activation status of pathways linked to cell cycle regulation

  • Examine effects on CDK/cyclin complex formation and activity

  • Investigate the impact on retinoblastoma protein phosphorylation

In vivo tumor models:

• Xenograft studies with controlled GMFB delivery

• Genetic models with inducible GMFB expression

• Histological and immunohistochemical assessment of proliferation markers

When investigating these properties, researchers should control for potential artifacts from the His tag and consider the physiological relevance of the concentrations being tested.

How can researchers accurately measure GMFB expression levels in tissue samples?

Accurate measurement of GMFB expression requires careful selection of appropriate techniques based on the research question:

Protein-level detection:

• Enzyme Immunoassay (EIA):

  • Two-site EIAs using specific antibodies have been developed with high sensitivity

  • These systems can detect GMFB in both human and rat samples

  • The assay allows quantification in various tissues and serum samples

• Western blotting:

  • Use validated antibodies against GMFB (not the His tag)

  • Include appropriate positive and negative controls

  • Implement densitometric analysis for semi-quantitative assessment

  • Consider using internal loading controls appropriate for the tissue type

• Mass spectrometry:

  • For absolute quantification, use targeted approaches like MRM/PRM

  • Include isotopically labeled peptide standards

  • Focus on unique peptides that distinguish GMFB from related proteins

mRNA-level detection:

• qRT-PCR:

  • Design primers specific to GMFB that don't amplify GMFG

  • Validate primer efficiency and specificity

  • Use appropriate reference genes for normalization

  • Consider absolute quantification with standard curves

• RNA-seq:

  • Apply appropriate normalization methods

  • Validate key findings with qRT-PCR

  • Account for potential splice variants

Tissue localization:

• Immunohistochemistry/Immunofluorescence:

  • Use validated antibodies with appropriate controls

  • Implement antigen retrieval if necessary

  • Consider dual labeling with cell-type specific markers

  • Use quantitative image analysis for expression level assessment

When designing studies to measure GMFB expression, researchers should be aware of:

• Potential cross-reactivity with GMFG

• Variations in expression across different brain regions

• The need to distinguish between intracellular and cell-surface GMFB

• The importance of standardized sampling and processing procedures