Activin A


Activin A is a TGF-β family member that exhibits a wide range of biological activities, including regulation of cellular proliferation and differentiation, and promotion of neuronal survival. Elevated levels of Activin A in human colorectal tumors and in postmenopausal women have been implicated in colorectal and breast cancers, respectively. The biological activities of Activin A can be neutralized by inhibins and by the diffusible TGF-β antagonist, follistatin. Activin A binds to the two forms of activin receptor type I (Act RI-A and Act RI-B) and two forms of activin receptor type II (Act RII-A and Act RII-B). Activins are homodimers or heterodimers of different β subunits. They are produced as precursor proteins with an amino terminal propeptide that is cleaved to release the C-terminal bioactive ligand. Recombinant Human/Murine/Rat Activin A is a 26.0 kDa disulfide-linked homodimer of two βA chains, each containing 116 amino acid residues.

Product

Catalog Nr

Size

Concentration


Pricing

human Activin A
P2282S
0.1 ml
1 mg/ml
human Activin A
P2282M
1 ml
1 mg/ml
human Activin A
P2282L
10 ml
1 mg/ml
human Activin A
P2282X
100 ml
1 mg/ml
human Activin A
P2282B
bulk

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Product Information
Product Literature & Protocol
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Product Information
Source: 
Human Activin A was improved through protein engineering efforts including directed evolution and rational design.  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.

Molecular Weight: 
~26 kDa

Purity: 
>98 % by SDS-PAGE and analysis by protein mass spectrometry

Sequence Analysis: 
Purified recombinant protein was subjected to peptide mass fingerprinting (PMF) by MALDI-TOF/TOF (PMF + MS/MS).

Cross Reactivity: 
Human, rhesus, cynomolgus/cercopithecine, mouse/murine, rat/murine, pig/porcine, cow/bovine, goat/caprine, dog/canine, cat/feline, horse/equine, frog/ranine

Biological Activity: 
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/neuregulinβ-1, LR3-IGF1 and recombinant 0.1% HSA. Activin A is supplemented at 20 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.  ED50 is ≤ 2.0 ng/ml, corresponding to a specific activity of ≥ 5 x 105 units/mg of recombinant human Activin A as determined by its ability to inhibit the proliferation of MPC-11 cells (transformed B lymphocytes from mouse strain BALB/c).

Endotoxin: 
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. 

Appearance: 
A sterile, aqueous, clear and colorless solution. 

Formulation: 
Human Activin A 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 7.4. The reconstituted solution has been filter sterilized by passing through a 0.22 micron PES membrane and tested to be negative for mycoplasma contamination. 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).

Storage/Stability: 
Human Activin A 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 FGF2-G3 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 but do not vortex.

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
0.03–0.2 pmols*
X μl
0.2–0.5 pmols**
X μl
10 μl
NovoHelix
HiFi DNA Assembly Master Mix
 10 μl10 μl
10 μl
 Deionized H2O10-X μl
 10-X μl0
 Total Volume20 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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Supporting Documents

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