Recombinant Mouse Kit ligand (Kitlg), partial (Active)

What is Recombinant Mouse Kit ligand and what are its primary research applications?

Recombinant Mouse Kit ligand, also known as Stem Cell Factor (SCF), c-kit ligand, mast cell growth factor (MGF), or steel factor (SLF), is a widely expressed 28-40 kDa type I transmembrane glycoprotein that promotes the survival, differentiation, and mobilization of multiple cell types . The protein is primarily used in research to:

• Stimulate proliferation of hematopoietic progenitor cells

• Study mast cell development and activation

• Investigate signaling pathways in cell differentiation models

• Examine hematopoietic stem cell (HSC) maintenance in bone marrow niches

The recombinant protein is typically derived from E. coli expression systems comprising amino acids Lys26-Ala189 of the mouse sequence, with an N-terminal Met .

How should Recombinant Mouse Kit ligand be reconstituted and stored for optimal activity?

Proper reconstitution and storage are critical for maintaining protein activity:

When selecting between formulations, consider that the carrier protein (BSA) enhances stability, increases shelf-life, and allows storage at more dilute concentrations . The carrier-free version is recommended for applications where BSA might interfere with experimental outcomes.

What bioassays confirm the activity of Recombinant Mouse Kit ligand preparations?

The biological activity of Recombinant Mouse Kit ligand can be verified through cell proliferation assays. The TF-1 human erythroleukemic cell line is commonly used as a standard bioassay system. In this application:

• Recombinant Mouse SCF stimulates proliferation in TF-1 cells in a dose-dependent manner

• The effective dose (ED50) typically ranges from 2.5-10 ng/mL

• Activity can be visualized as a dose-response curve

• Protein purity can be confirmed via SDS-PAGE under reducing conditions, where Kit ligand appears as a single band at approximately 19 kDa

These standardized assays ensure batch-to-batch consistency and biological relevance of the recombinant protein.

How do membrane-bound and soluble forms of Kit ligand differentially regulate hematopoietic stem cells?

Recent research has clarified the distinct roles of membrane-bound Kit ligand (mKITL) versus soluble Kit ligand (sKITL) in HSC regulation:

• Systemic sKITL appears to be the primary regulator of HSC maintenance, rather than local membrane-bound expression

• Deletion of endothelial-derived mKITL (using Tie2ΔEx7 mice) reduces circulating sKITL levels by approximately 26% without affecting bone marrow sKITL levels

• Importantly, this specific deletion of mKITL does not reduce bone marrow cellularity, myelo-erythroid progenitor cells, or phenotypically and functionally defined HSCs

• This contradicts earlier interpretations that suggested mKITL acts locally in creating bone marrow HSC niches

These findings highlight that experimental designs targeting Kit ligand should consider both local and systemic effects, particularly when using conditional knockout approaches.

What methodological approaches can differentiate between systemic and niche-specific effects of Kit ligand?

Distinguishing between systemic and niche-specific effects requires sophisticated experimental approaches:

• Conditional deletion models: Using Cre-loxP systems with tissue-specific promoters (e.g., Tie2-Cre for endothelial cells)

• Bone marrow transplantation assays: Implanting Kit ligand-deleted bones into mice with normal systemic Kit ligand levels

• Measurement of both local and circulating Kit ligand: Quantifying Kit ligand in both bone marrow and blood simultaneously

• Complementation experiments: Restoring systemic Kit ligand levels in knockout models

Recent studies demonstrate that when Kit ligand-deleted bones are implanted into mice with normal systemic sKITL levels, HSCs remain unaffected, supporting the hypothesis that systemic rather than local niche expression of sKITL regulates HSCs .

How can neutralization assays be designed to study Kit ligand function?

Neutralization assays provide critical insights into Kit ligand functionality:

The Neutralization Dose (ND50) approach measures the ability of antibodies to block Kit ligand activity:

• Recombinant Mouse SCF/c-kit Ligand (typically at 25 ng/mL) is used to stimulate proliferation in TF-1 cells

• Increasing concentrations of Goat Anti-Mouse SCF/c-kit Ligand Antigen Affinity-purified Polyclonal Antibody are added

• The ND50 (concentration required for 50% neutralization) is typically 0.3-0.6 μg/mL

• Results can be visualized as a neutralization curve showing dose-dependent inhibition

This assay design allows researchers to quantitatively assess both agonists and antagonists of Kit ligand signaling pathways.

What are the current challenges in interpreting Kit ligand knockout studies?

Interpreting Kit ligand knockout studies presents several methodological challenges:

• Systemic effects confounding local deletion: Endothelial-specific deletion of Kit ligand reduces systemic levels, complicating interpretation of niche-specific effects

• Developmental timing: Deletion effects may vary between embryonic, adolescent, and adult stages

• Multiple cell type expression: Kit ligand is expressed by various cell types beyond endothelial cells, including perivascular cells

• Functional redundancy: Other cytokines may compensate for Kit ligand deletion in certain contexts

• Isoform-specific functions: Distinguishing the specific roles of membrane-bound versus soluble forms requires careful experimental design

Recent studies highlight that "knocking out key regulatory molecules expressed in candidate BM HSC niche cells" as an approach to establish direct regulatory roles requires careful consideration of systemic effects that may confound interpretation .

What cell types and developmental processes can be studied using Recombinant Mouse Kit ligand?

Kit ligand influences multiple cell lineages and developmental processes:

• Hematopoietic lineages: Myeloid, erythroid, megakaryocytic, and lymphoid progenitors

• Non-hematopoietic cells: Germ cells, melanocyte progenitors

• Immune cells: Primary growth and activation factor for mast cells and eosinophils

• Cardiac recovery: Assists in recovery of cardiac function following myocardial infarction by increasing cardiomyocyte numbers and vascular channels

Each application requires optimization of Kit ligand concentration and treatment duration based on the specific cell type and experimental endpoint.

How can signaling mechanisms of Kit ligand be studied in research settings?

Studying Kit ligand signaling mechanisms involves several methodological approaches:

• Receptor dimerization assays: Non-covalent dimers of transmembrane or soluble SCF interact with SCF R/c-kit to trigger receptor dimerization

• Phosphorylation studies: Measuring downstream phosphorylation events following Kit receptor activation

• Transcriptional profiling: Identifying gene expression changes induced by Kit ligand signaling

• Mutant receptor studies: Using c-kit receptor mutants to dissect signaling pathway components

• Pathway inhibition: Combining Kit ligand with specific pathway inhibitors to identify critical nodes

Notably, in signaling studies, the differences between human and mouse Kit ligand should be considered, as rat SCF is active on both mouse and human cells, but human SCF is only weakly active on mouse cells .

What quality control parameters should be assessed when working with Recombinant Mouse Kit ligand?

Critical quality control parameters include:

Each laboratory should determine optimal dilutions for specific applications, as effectiveness can vary based on experimental conditions .

How is Kit ligand being utilized in hematological malignancy research?

Kit ligand plays significant roles in hematological malignancy research:

• Used in studies examining MLL-fusion leukemia development mechanisms

• Applied in models investigating TLR-VCAM1 pathway activation

• Employed in research on IMPDH inhibition effects on leukemia progression

• Utilized to understand the development and expansion of erythroleukemic cell lines

These applications facilitate understanding of both normal hematopoiesis and pathological conditions involving dysregulated Kit signaling.

What emerging therapeutic strategies target Kit ligand pathways?

Novel therapeutic approaches targeting Kit ligand pathways include:

• Electroacupuncture-based modulation: Studies show electroacupuncture at specific points (e.g., ST36) can increase bone marrow-derived interstitial cells of Cajal via SDF-1/CXCR4 and mSCF/Kit-ETV1 pathways

• IMPDH inhibition: This approach activates TLR-VCAM1 pathway and suppresses MLL-fusion leukemia development

• Cytokine/chemokine response pattern modulation: Radiation responses can be modified through Kit ligand pathway targeting

• Wnt/β-catenin signaling interaction: Epiblast-like stem cells established by Wnt/β-catenin signaling show distinct features in formative pluripotency and germline competence related to Kit signaling

These emerging approaches highlight the therapeutic potential of targeting Kit ligand pathways in various disease contexts.