NRG1 — Neuregulin β-1
Neuregulin/Heregulin is a family of structurally-related polypeptide growth factors derived from alternatively spliced genes (NRG1, NRG2, NRG3 and NRG4). To date, there are over 14 soluble and transmembrane proteins derived from the NRG1 gene. Proteolytic processing of the extracellular domain of the transmembrane NRG1 isoforms releases soluble growth factors. HRG1-β1 contains an Ig domain and an EGF-like domain; the latter is necessary for direct binding to receptor tyrosine kinases erb3 and erb4. This binding induces erb3 and erb4 heterodimerization with erb2, stimulating intrinsic kinase activity that leads to tyrosine phosphorylation. Although HRG1-β1's biological effects are still unclear, it has been found to promote motility and invasiveness of breast cancer cells, which may also involve up-regulation of expression and function of the autocrine motility-promoting factor (AMF). Recombinant human Neuregulinβ-1 (NRG1-B1/HRG1-B1) is a 7.5 kDa polypeptide consisting of only the EGF domain of neuregulinβ-1 (65 amino acid residues).
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Product Literature & Protocol
FAQs & Troubleshooting
Human NRG1/neuregulinβ-1 was improved through protein engineering efforts. The final engineered protein was synthesized as a codon-optimized minigene, was expressed heterologously in an E. coli strain with the assistance of chaperone proteins and was purified by proprietary chromatographic techniques. This product is manufactured in the USA using chemically-defined components and, therefore, does not contain manufacturing components such as serum or animal-derived raw materials. The recombinant protein was tested and normalized to standard activity assays for growth of human pluripotent stem cells.
>98 % by SDS-PAGE and analysis by protein mass spectrometry
Purified recombinant protein was subjected to peptide mass fingerprinting (PMF) by MALDI-TOF/TOF (PMF + MS/MS).
Proliferation of a human pluripotent stem cell (hPSC) line cultured under 5% oxygen tension & 5% CO2 on a recombinant laminin 521 matrix with a serum-free DMEM/F12 standard basal media containing FGF2-G3, TGFβ, transferrin, vitamin C (L-ascorbic acid-2-phosphate), NRG1 (neuregulin-1β), LR3-IGF1 and recombinant 0.1% HSA. NRG1 is supplemented at 0.1 ng/ml. The hPSC line contains OCT4-P2A-tdTomato knock-in at the human POU5F1 locus to indicate which colonies are still pluripotent. Cells were routinely passaged at low seeding densities to monitor proliferation of hPSC growth for 5 passages and viability assessed by PrestoBlue. The population doubling level (PDL) was calculated by the formula PDL = 3.32[log10(n/n0)], where n = cell number and n0 = number of cells seeded.
The specific activity of recombinant human NRG1 is ≥ 2 x 106 units/mg as determined by the dose-dependent proliferation of human MCF-7 cells where ED50 ≤ 0.5 ng/ml.
Lipopolysaccharide (LPS) was determined by the standard LAL (Limulus amebocyte lysate) test to be < 0.5 EU/µg. Due to potential environmental sustainability concerns (collection of the hemolymph used in pharmaceutical testing may negatively affect horseshoe crab populations), a quantitative and more sensitive endotoxin assay was developed using a pyrogen‐testing cell model with knock-in (targeted, single-copy integration) of TLR4/CD14/MD2 at the safe harbor locus AAVS1/PPP1R12C on human chromosome 19. The TLR4/CD14/MD2 assay is used as an orthogonal screen for LPS in lieu of the LAL method and has a detection limit of 0.005 EU/ml.
A sterile, aqueous, clear and colorless solution.
Human NRG1 is supplied reconstituted at 1 mg/ml in a stabilization buffer containing 9 mg/mL benzyl alcohol, 50 mM sodium phosphate, 250 mM sodium chloride, 2 mg/mL polysorbate 20, 5% trehalose, 10% v/v glycerol at a ~ pH 7.2. Each milliliter (mL) contains 0.9% benzyl alcohol added as a bacteriostatic preservative. Polysorbate 20 is a nonionic surfactant commonly used in the formulation of protein biologics to prevent protein aggregation and denaturation (protein unfolding).
Human NRG1 is supplied at 1 mg/ml in stabilization buffer and remains bioactive for 3 weeks if maintained refrigerated 2º to 8ºC (35º to 46ºF). Stock solutions of human NRG1 in its concentrated form can be stored at least 3 months at -20°C and at least 12 months at -80°C from the date of manufacture with no loss of activity on ES cells. For long term storage, add 0.1% w/v carrier protein such as recombinant human serum albumin (HSA) and avoid repeated freeze-thaw cycles by aliquoting. Multiple freeze-thaw cycles reduce potency and, therefore, aliquoting working stocks is strongly recommended. To maximize recovery, spin the microcentrifuge tube prior to opening.
Disclaimer & Precautions:
This product is solely for research and development use only and may be subject to conditional use and licensing restrictions. The product shall not be used as an advanced pharmaceutical intermediate (API) or investigational drug or a biologic. This product is not intended to be used as a therapeutic agent or facilitate clinical diagnosis or be used as an in vitro diagnostic (IVD) product.
The Food and Drug Administration (FDA) and Center for Biologics Evaluation and Research (CBER) define an IVD as:
“In vitro diagnostic products are those reagents, instruments, and systems intended for use in the diagnosis of disease or other conditions, including a determination of the state of health, in order to cure, mitigate, treat, or prevent disease or its sequelae. Such products are intended for use in the collection, preparation, and examination of specimens taken from the human body. These products are devices as defined in section 201(h)of the Federal Food, Drug, and Cosmetic Act (the act), and may also be biological products subject to section 351 of the Public Health Service Act. Title 21, Code of Federal Regulations (CFR), section 809.3(a).”
This product shall not be used or formulated in any agricultural, pesticidal, veterinary or animal products, food additives or household chemicals or any other unspecified use. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices. NovoHelix distributes products for basic and translational research use only. NovoHelix will report any unspecified use to respective regulatory authorities for enforcement to ensure safeguarding of our research products from potential abuse.
Notice to purchaser:
The purchase price of this product includes a limited, non-transferable license under U.S. and foreign patents or applications owned by NovoHelix to use this product. No other license under these patents or applications is conveyed expressly or by implication by purchase of this product.
Product Literature & Protocol
- HiFi DNA Assembly Protocol
Recommended Amount of Fragments Used for Assembly
|2–3 Fragment Assembly*||4–6 Fragment Assembly**||Positive Control✝|
|Recommended DNA Molar Ratio||vector:insert = 1:2||vector:insert = 1:1|
|Total Amount of Fragments|
HiFi DNA Assembly Master Mix
|10 μl||10 μl||10 μl|
|Deionized H2O||10-X μl||10-X μl||0|
|Total Volume||20 μl✝✝||20 μl✝✝||20 μl|
* Optimized cloning efficiency is 50–100 ng of vector with 2-fold excess of inserts.
Use 5 times more insert if size is less than 200 bp. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20%.
**To achieve optimal assembly efficiency, design ≥ 20 bp overlap regions between each fragment with equimolarity (suggested: 0.05 pmol each).
† Control reagents are provided for 5 experiments.
†† If greater numbers of fragments are assembled, increase the volume of the reaction linearly by using additional NovoHelix HiFi DNA Assembly Master Mix. Alternatively, pool the DNA fragments into an equimolar mix first and then re-purify these pooled equimolar fragments over a micro-column and elute with a minimum volume (~10-µl). The eluate may be reapplied to the same micro-column membrane to improve elution of large DNA fragments without increasing the final volume..
Recommended Storage Condition:
This assembly mixture can be stored at -20 °C for at least one year. The enzymes remain active following at least 10 freeze-thaw cycles.
FAQs & Troubleshooting
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