Recombinant Proteins Support—Getting Started
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Recombinant protein handling
Lyophilized proteins can typically be stored at 2–8°C for several weeks, or stored desiccated at –20°C for long-term storage.
Protein solutions are generally not very stable when frozen at low concentration. Upon freeze and thaw, some proteins in the solution may stick to the wall of the container, which results in significant reduction of protein concentration if the starting concentration was low. Therefore, carrier proteins are used to reduce such loss. The most commonly used carrier proteins include bovine serum albumin (BSA), human serum albumin (HSA), or fetal bovine serum (FBS). These carrier proteins are generally used at 0.1% concentration. As a rule of thumb, if the concentration of the recombinant protein is less than 0.5 mg/mL, it is a good idea to add some carrier protein.
The instructions for reconstituting the lyophilized protein are provided on the data sheet. We recommend that the container be first centrifuged to concentrate the powder at the bottom of the tube. Most proteins can be reconstituted with the addition of sterile, distilled water. For 100 μg of protein, the amount of water that should be added should be between 100 µL and 1 mL, resulting in a protein solution with a concentration of between 1 mg/mL and 0.1 mg/mL. The product analysis sheet will indicate when a diluent other than water is required.
The reconstituted protein should be apportioned into working aliquots. A useful size for a working aliquot is 10 or 20 μL. The aliquots should be stored in polypropylene tubes. Polypropylene is great for this application because proteins do not stick to this material very well. Proteins may adhere to glass or polystyrene, and we suggest avoiding the use of storage containers made of these materials. The aliquots should be stored at –20°C. If you have access to a –80°C freezer, storage at this temperature is even better.
Repeated freeze/thaws will affect the stability of the recombinant protein. For example, freezing will significantly affect the pH of the protein solution and might cause denaturation of the protein (Arch Biochem Biophys 384:398 (2000)).
The recombinant proteins provided by Thermo Fisher Scientific™ are usually produced in different expression systems such as E. coli, insect cells, or mammalian cells. The major differences in recombinant proteins produced in different expression systems are in the post-translational modifications present, such as glycosylation. Recombinant proteins produced in E. coli are not glycosylated. Recombinant proteins produced in insect cells are partially glycosylated without galactose and sialic acid and not branched. Recombinant proteins produced in mammalian cells are fully glycosylated.
Note: Mimic™ Sf9 Insect Cells (a derivative of the Sf9 insect cell line that has been modified to stably express a variety of mammalian glycosyltransferases) can be used for production of complex N-glycans with terminal sialic acid and galactose.
In most of the cases, glycosylation of a growth factor or cytokine does not affect how it binds to a receptor directly. So the biological activity of a recombinant growth factor or cytokine is not significantly affected by glycosylation within an in vitro study. However, the glycosylated protein is usually less sensitive to protease degradation and exhibits much longer half life in vivo than the same protein without glycosylation. Therefore, for in vivo studies selecting recombinant protein produced in a mammalian expression system or insect expression system might be a better choice than the same recombinant protein produced in E. coli.
Bioassays are intended to measure the biological activity of a given growth factor or cytokine. In most of the cases, the bioassays are cell-based tests using different indicator cells such as primary cells or cell lines. The most commonly used bioassays include cell proliferation assay, chemotaxis assay, cytokine production assay, and cytotoxicity assay. The biological activity of a given cytokine is expressed as ED50, which represents the concentration of the cytokine that induces 50% of the maximum response. This method of expressing potency should only be used for cytokines whose dose-response curves are sigmoidal in shape.
The ED50 is defined as the cytokine concentration at which the activity is 50% of the maximum response. This method of expressing potency should only be used for cytokines whose dose-response curves are sigmoidal in shape. The formula for converting the activity as an ED50 in ng/mL (NOTE: if in pg/mL, convert to ng/mL) to specific activity in units/mg is (where there is an ED50 expressed as a range, choose the mid-point in the range):
1 x 10E6/ED50* (ng/mL) = specific activity in Units/mg of protein
No, the biological activities of recombinant growth factors and cytokines provided by Thermo Fisher Scientific™ have not been calibrated using the WHO standard. Therefore, the activity (unit/mg of protein) converted from ED50 is applicable to the indicator cells used in the QC assay (the information on the indicator cells used for each recombinant growth factor or cytokine is available in the product insert). Therefore, the biological activity of a particular recombinant protein from a different vendor can only be compared when the same indicator cells are used.
Embryonic stem (ES) cells are derived from the early mammalian embryo and are capable of unlimited, undifferentiated proliferation in vitro while maintaining their potential to differentiate into a wide of range of adult tissues including germ cells. The pluripotency of the ES cells is normally demonstrated in vitro by inducing ES cells to differentiate into embryoid bodies and checking lineage-specific markers for differentiated cells in three body layers (endo, meso, and ectoderm), or injecting them into immunodeficient mice and determining the cell types produced in the teratomas.
Human ES cells are typically maintained on top of irradiated mouse embryonic fibroblast cells (MEF) with high-glucose DMEM supplemented with 20% FBS (or 20% of Gibco™ KnockOut™ Serum Replacement), 0.1 mM β-mercaptoethanol, 1% of nonessential amino acids (Science 282:1145 (1998); Dev Biol 227:271 (2000)).
Human ES cells can also be maintained without feeder cells on Matrigel™ matrix–coated culture vessels in medium conditioned by MEF (Nat Med 10:55 (2004)). Once the ES cells are established on the MEF or MEF-conditioned medium, these cells may be maintained under feeder-free conditions using Gibco™ Geltrex™ Matrix–coated culture vessels in Gibco™ StemPro™ SFM supplemented with bFGF and β-mercaptoethanol or Gibco™ Essential 8™ Medium and vitronectin-coated vessels.
Human ES cells are generally characterized by their typical morphology (they grow as tightly packed clusters of small cells with high ratio of nucleus to cytoplasm); surface marker expression; RT-PCR detection of stem cell–specific gene expression (such as Oct3/4, Sox2, and Nanog); alkaline phosphatase staining, and telomerase activity assay. The most commonly used ES specific surface markers include stage-specific embryonic antigens SSEA-3 and SSEA-4 for human ES cells. Other ES-specific surface antigens also include TRA-1-60 and TRA-1-81. (Science 282:1145 (1998).
For human ES cell differentiation: BMP-4 (Cat. No. PHC9533), EGF (Cat. No. PHG0311), and HGF (Cat. No. PHG0324) (Proc Natl Acad Sci U S A 97:11307 (2000)).
The primary translation product for bFGF is composed of 155 amino acids without a traditional signal peptide (EMBO J 5:2523 (1986)). The mature form of secreted human bFGF is 146 amino acids long (nine amino acid residues at the N-terminus are likely proteolytically cleaved from the 155-aa precursor) ((FEBS Lett 185:177 (1985)). The biological activities of our 155 aa form (Cat. No. PHG0264) and 146 aa form (Cat. No. PHG0024) are very similar when tested under the same conditions, indicating that this N terminal region of bFGF is not involved either in biological activity or in binding to FGF cell surface receptors. Although there is no difference in biological function for these two forms, we recommended the 155 aa form for ES cell use and the 146 aa form for neural cells study. This recommendation is purely based on how we used both forms in house during product development (i.e., the 155 aa form was used in ES cell study and the 146 aa form was used for neural science study), and not on product performance differences observed at the time.
Induced pluripotent stem cells (iPS or iPSCs) are pluripotent stem cells directly generated by introducing combination of genes coding for “reprogramming factors” into adult cells. These reprogramming factors include Oct4, Sox2, c-Myc, KLF4, NANOG, and LIN28. Yu, et al, generated iPS from a human mesenchymal cell line using lentiviral vectors carrying Oct4, Sox2, NANOG, and LIN28 genes (Science 318:1917 (2007)). Using a similar approach, Takahashi et al, generated iPS from human primary fibroblast cells by introducing genes coding for Oct3, Sox2, KLF4, and c-Myc into these cells (Cell 131:861 (2007)). iPS generated by reprogramming are similar to human ES cells in morphology, the capacity for unlimited proliferation, surface-antigen expression, gene expression, the ability to differentiate into cell types representing the three germ layers in vitro, and the ability to form teratomas after injection into SCID mice.
The growth conditions for human iPS are identical to human ES cells. Like human ES cells, the human iPS cells can grow on irradiated MEF feeder cells in DMEM/F12 with 20% KnockOut™ SR, 0.1 mM NEAA, 1 mM glutamine, 0.1 mM b-ME, with 100 ng/mL bFGF or on a Matrigel™ Matrix–coated dish with MEF-conditioned medium with 100 ng/mL bFGF (Science 318:1917 (2007)). Both Human ES cells and iPS cells can be grown in KnockOut™ ESC/iPS medium (Cat. No. A14131).
The same growth factors used for ES cell study can also be used for iPS study:
For human ES cell differentiation: BMP-4 (Cat. No. PHC9533), EGF (Cat. No. PHG0311), and HGF (Cat. No. PHG0324) (Proc Natl Acad Sci U S A 97:11307 (2000)).
Mesenchymal stem cells (MSCs) are multipotent cells isolated primarily from bone marrow or fat tissues that exhibit the ability to differentiate into bone, cartilage, and fat cells. Under normal cell culture conditions, MSCs isolated from bone marrow are spindle shaped with the unique ability to adhere to uncoated plastic culture dishes (Arthritis Res Ther 9:204 (2007)).
MSCs may be cultured in either serum-containing medium or serum-free medium. The standard culturing conditions are DMEM, low glucose with 10% FBS. MSCs can also be grown in reduced-serum (2%) Gibco™ MesenPRO RS™ Medium (Cat. No. 12746-012), or in serum-free Gibco™ StemPro™ MSC SFM XenoFree Medium (Cat. No. A10675) and Gibco™ StemPro™ MSC SFM (Cat. No. A10332). In general, MSCs grow better under hypoxic conditions (2% O2).
Please see our suggestions below for inducing MSC differentiation:
Differentiation conditions for MSCs maintained with StemPro™ MSC medium: Adipogenic differentiation seed cells in 1.24 x 10E5 cells/cm2 in low glucose DMEM with 10% MSC qualified FBS (Cat. No. 12662011), 2 mM glutamax, 5 µg/mL gentamicin, and 58 µg/mL human recombinant insulin, 1 µM desamethason, 0.5 mM isobutyl-methylxanthine, and 200 µM indomethacin.
Osteogenic differentiation: seed cells in 3.08 x 10E4 cells/cm2 in low glucose DMEM with MSC qualified FBS, 2 mM glutamax, 5 µg/mL gentamicin, 10 mM glycerol-2-phosphate, 50 µM L-ascorbic acid, 10 ng/mL BMP-2, 100 nM dexamethasone.
Chondrogenic differentiation: seed cells in 10 µL drops (8 x 10E4 cells/drop); after 2 hours’ attachment feed cells in low glucose DMEM with 10% MSC qualified FBS, 2 mM glutamax, 5 µg/mL gentamicin, 6.25 µg/mL recombinant human insulin, 10 ng/mL recombinant human TGF-β1, 50 nM L-ascorbic acid (Blood 112:295 (2008)).
Alternatively we offer these differentiation kits: StemPro™ Adipogenesis Differentiation Kit (Cat. No. A10070-01), StemPro™ Osteogenesis Differentiation Kit (Cat. No. A10072-01), and StemPro™ Chondrogenensis Differentiation Kit (Cat. No. 10071-01).
Growth factors used for MSC maintenance and self-renewal: LIF (PHC9484), bFGF (PHG0261), EGF (PHG0311), HGF (PHG0324), PDGF (PHG0043), and Wnt3.
Growth factors or cytokines for MSC differentiation: BMP-2 (PHC7145), and TGF-β1(PHG9211).
Neural stem cells (NSCs) are self-renewing multipotent cells of the nervous system capable of differentiating into neurons, oligodendrocytes, and astrocytes. NSC can be generated by induced differentiation from embryonic stem (ES) cells, or isolated from various regions of the brain including the cortex, the subventricular zone (SVZ), and the ventricular zone, or generated from bone marrow–derived mesenchymal stem cells (MSCs) (J Cell Biochem 114:764 (2013)). NSCs are valuable tools for the study of neurogenesis and neurotransmitter and receptor function. NSCs were used in the investigation of different CNS disorders such as PD and Huntington’s disease in various animal models (J Cell Biochem 114:764 (2013)).
NSCs are generally characterized by their ability to form neurospheres when plated at cloning density (Nat Methods 2:333 (2005)). NSCs can also be characterized by (1) RT-PCR of Sox1, Sox2, and Nestin or (2) immunohistochemical staining for nestin, Pax6, Sox2, and Ki67.
Human NSCs can grow in Gibco™ StemPro™ NSC SFM (Cat. No. A1050901) on dishes pre-coated with Gibco™ Geltrex™ Matrix or Gibco™ CELLstart™ substrate. Alternatively, if the goal is to obtain neurons, NSCs can also be grown on Neurobasal™ medium supplemented with Gibco™ B-27™ supplements without vitamin A on a pre-coated dish.
For NSC expansion, the following growth factors are used: recombinant EGF (Cat. No. PHG0314), recombinant bFGF (Cat. No. PHG0024), and recombinant VEGF (Cat. No. PHC9394). In addition, several neurotrophins such as BDNF (Cat. No. 10908010), CNTF (Cat. No. PHC7015), and GDNF (Cat. No. PHC7044) are also used in the related studies.
Historically we have provided high-quality Gibco™ recombinant proteins, mainly focusing on the major growth factors, cytokines, and chemokines commonly used in cell culture. To expand our recombinant protein portfolio, we have partnered with Sino Biological to offer around 1,500 recombinant proteins (co-branded Thermo Fisher Scientific and Sino Biological). Sino recombinant proteins offer four major advantages: (1) wide selection of recombinant proteins including adhesion proteins, cytokines, chemokines, growth factors, influenza proteins, proteases, phosphatases, transcription factors, channel proteins, and receptors; (2) competitive pricing; (3) broader size selections for each protein and finally (4) majority are expressed in mammalian cells, with 90% of them are expressed in human cell lines to help ensure the presence of the correct mammalian post-translational modifications.
Since Sino recombinant proteins are usually lyophilized in a buffered solution, we generally recommend that you reconstitute in double-distilled water at a concentration of 0.2 mg/mL (unless specifically instructed to reconstitute in another buffers). Store the stock solution at –20°C in aliquots.
Sino recombinant proteins are provided as lyophilized powder, and we recommend storing themt at –20°C. The shelf life is one year from receipt if the product is stored as recommended.
Since 90% of the Sino recombinant proteins are produced in human cells as secreted proteins it is very likely they retain native conformation and biological activity. This is especially true for those extracellular secreted proteins such as growth factors, cytokines, chemokines, and secreted enzymes. For some Sino recombinant proteins, biological activity is tested using a cell-based assay or functional ELISA assay, and ED50 values are provided on the product page.
Functional ELISA is an in vitro ligand binding–based assay performed in the ELISA format. It tests the capability of a target protein to bind to its native binding partner i.e., a growth factor to its receptor. If a growth factor binds to its native receptor in this assay, it is highly likely the growth factor is functional. The ED50 is the concentration of growth factor (on a receptor-coated plate) that gives half of the maximum signal in an OD reading.
Although in both cases, a lower ED50 indicates higher biological activity, these two values can’t be directly converted. Because the ED50 from a functional ELISA indicates the concentration of a given growth factor or cytokine that gives half maximum binding to its receptors, this is not necessarily the concentration that gives half maximal biological response in the target cell. In addition, the ED50 of the cell-based assay varies significantly depending on the indicator cells used. ED50 values in a functional ELISA do not change that much.
Some Sino recombinant proteins are supplied without biological activity data. In most of the cases, that’s an indication that the biological activity test was not carried out; it does not necessarily mean the protein is inactive. In the absence of biological activity data, the applications for which these proteins are validated are as protein molecular weight standards, western blot positive controls (if the recombinant protein is in full length), or immunogens. Other possible uses for these untested proteins are as ligand binding assay or ELISA standards.
For Research Use Only. Not for use in diagnostic procedures.