Recombinant Human Angiogenin

Recombinant Human Angiogenin is used in research because it is a multifunctional protein with critical roles in angiogenesis, tissue regeneration, neuroprotection, inflammation modulation, and tumor biology.

Key scientific applications and rationale include:

• Angiogenesis studies: Angiogenin is a potent inducer of new blood vessel formation, making it essential for modeling and dissecting mechanisms of angiogenesis in vitro and in vivo. It is particularly relevant in cancer research, wound healing, and tissue engineering, where vascularization is a key process.

• Tissue regeneration and wound healing: Recombinant angiogenin stimulates collagen fiber formation, fibroblast proliferation, and dermal thickening, all of which are important for studying skin repair, tissue remodeling, and regenerative medicine applications.

• Neuroprotection and neurodegenerative disease models: Angiogenin has demonstrated neuroprotective effects, such as improving motor function and extending lifespan in ALS mouse models, and protecting neurons in models of Parkinson’s disease. It is used to investigate mechanisms of neuronal survival and potential therapeutic strategies for neurodegenerative disorders.

• Inflammation and immune modulation: Angiogenin regulates both pro- and anti-inflammatory cytokine production, making it valuable for studying immune responses, inflammation, and their resolution in various disease models.

• Cancer biology: Angiogenin is implicated in tumor growth, metastasis, and survival, particularly in gastrointestinal cancers. It is used to study tumor angiogenesis, cancer cell survival, and as a potential therapeutic target or biomarker.

• Molecular and cellular mechanisms: Recombinant angiogenin enables detailed studies of its RNase activity, nuclear translocation, effects on rRNA processing, chromatin remodeling, and signaling pathways (e.g., PI3K/Akt/mTOR), which are relevant for understanding cell growth, apoptosis, and gene regulation.

• Diagnostic and therapeutic research: Variations in angiogenin levels are being explored as diagnostic or prognostic biomarkers for cancer, cardiovascular, and inflammatory diseases, and recombinant protein is essential for assay development and validation.

Using recombinant human angiogenin ensures consistency, purity, and reproducibility in experimental systems, which is critical for mechanistic studies, drug screening, and translational research.

Recombinant Human Angiogenin can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated and matched to your assay system. Recombinant proteins are commonly used as standards in ELISA kits for quantifying target analytes, including angiogenin, in biological samples.

Key considerations for use as an ELISA standard:

• Purity and Formulation: The recombinant angiogenin should be of high purity and, ideally, formulated in a buffer compatible with your assay. Some protocols recommend using recombinant protein with a carrier (e.g., BSA) for stability, especially in cell culture or as an ELISA standard.
• Calibration and Validation: Standards must be calibrated against a mass-calibrated reference to ensure accurate quantification. ELISA kits typically provide pre-calibrated standards, and if you use a recombinant protein not supplied with your kit, you must validate its concentration and performance in your assay system.
• Assay Compatibility: The recombinant standard should be recognized by the antibodies used in your ELISA. Most commercial ELISA kits for human angiogenin use recombinant human angiogenin as the standard for generating the calibration curve.
• Precision and Recovery: Validation studies show that recombinant standards can provide precise and accurate quantification when properly validated, with intra- and inter-assay coefficients of variation typically below 15%.

Best Practices:

• Prepare a fresh standard curve for each assay using serial dilutions of the recombinant angiogenin.
• Validate the recombinant standard in your specific assay matrix (e.g., serum, plasma, cell culture supernatant) to confirm recovery and linearity.
• If switching batches or sources of recombinant angiogenin, perform batch-to-batch validation to ensure consistency.

Limitations:

• Not all recombinant proteins are calibrated for use as ELISA standards; calibration against a master calibrator is necessary for quantitative accuracy.
• Carrier-free recombinant proteins may be less stable and require careful handling.

In summary, recombinant human angiogenin is suitable as an ELISA standard if you validate its concentration, compatibility, and performance within your assay system. This approach is standard practice in quantitative ELISA protocols for angiogenin and other biomarkers.

Recombinant Human Angiogenin has been validated in published research for several key applications, primarily in studies of angiogenesis, tissue regeneration, inflammation, neuroprotection, and cancer biology.

Key validated applications include:

• Angiogenesis assays: Recombinant human angiogenin is widely used to stimulate and assess new blood vessel formation in vitro and in vivo, confirming its role as a potent angiogenic factor.
• Tissue regeneration and wound healing models: It has been validated in animal models (e.g., intracutaneous injection in rats) to promote collagen fiber formation, increase fibroblast proliferation, and enhance dermal blood vessel density, supporting its use in regenerative medicine research.
• Inflammation studies: Angiogenin has been used to modulate cytokine production in human whole blood cells ex vivo, demonstrating both pro- and anti-inflammatory effects. It has also been shown to suppress leukocyte degranulation and modulate inflammatory cytokine expression in animal models.
• Neuroprotection: Recombinant angiogenin has been validated in neurodegenerative disease models (e.g., ALS and Parkinson’s disease in mice), where it reduces neuronal cell death and mortality, supporting its neuroprotective properties.
• Cancer research: It is used to study tumor angiogenesis, cancer cell survival, and metastasis, particularly in gastrointestinal and prostate cancer models. Angiogenin’s role in tumor growth, chromatin remodeling, and cell signaling pathways (e.g., PI3K/Akt) has been validated in vitro and in vivo.
• Cell signaling and gene regulation: Studies have validated its use in investigating ribonuclease activity, tRNA cleavage, and downstream effects on cell survival, apoptosis, and gene expression.

Additional validated uses include:

• Histological and morphological analyses of tissue samples following angiogenin treatment.
• Cytokine profiling in human blood or tissue culture systems to assess immune modulation.
• Cell proliferation and migration assays in endothelial and cancer cell lines.

These applications are supported by a range of experimental systems, including animal models, primary human cells, and established cell lines, with protocols involving direct protein administration, ex vivo stimulation, and molecular assays.

Reconstitution of Recombinant Human Angiogenin

Recombinant human angiogenin is typically supplied as a lyophilized powder and requires proper reconstitution before use in cell culture experiments. The reconstitution process is critical for maintaining protein functionality and stability.

Initial Reconstitution Steps

Begin by centrifuging the vial briefly before opening to concentrate the lyophilized powder at the bottom of the tube. This prevents loss of material during opening. Avoid vigorous vortexing or pipetting, as these actions can cause foaming and protein denaturation.

Dissolve the lyophilized protein in an appropriate diluent. The standard reconstitution medium is sterile phosphate-buffered saline (PBS) or double-distilled water (ddH₂O). The recommended reconstitution concentration is 100 µg/mL, and it is not advisable to reconstitute to concentrations below this threshold.

Allow the vial to reconstitute for 15-30 minutes at room temperature with gentle agitation to ensure complete dissolution.

Carrier Protein Addition

For long-term storage and enhanced stability, add a carrier protein such as human or bovine serum albumin (BSA) at a concentration of at least 0.1%. This protectant helps prevent protein aggregation and degradation during storage.

Storage and Stability Considerations

Short-term Storage

Reconstituted protein solutions can be stored at 4-8°C for 2-7 days. This timeframe is suitable for experiments planned within a week of reconstitution.

Long-term Storage

For extended storage, prepare aliquots of reconstituted samples and store at -20°C or below, where they remain stable for approximately 3 months. Lyophilized protein in its original form is generally stable for up to 12 months when stored at -20 to -80°C.

Critical consideration: Use a manual defrost freezer and avoid repeated freeze-thaw cycles, as these can compromise protein integrity and biological activity.

Preparation for Cell Culture Applications

When preparing angiogenin for hematopoietic cell culture studies, the protein can be added directly to culture media at predetermined concentrations. Culture periods typically range from 2 hours to 7 days or longer, depending on experimental objectives and the specific hematopoietic cell populations being studied.

For functional assays requiring precise concentration gradients, prepare serial dilutions from the 100 µg/mL stock solution using an appropriate dilution buffer to achieve the desired working concentrations.