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Optimize your experiments to get the best results. We’ve compiled a detailed knowledgebase of the top tips and tricks to meet your research needs related to reconstitution, storage, and handling of recombinant proteins (including Gibco PeproTech proteins) and their use in stem cell culture.
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Depending upon the product, the instructions for reconstituting the lyophilized protein are provided in either the product manual, product data sheet, or Certificate of Analysis (COA), which can be found on the product page. 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. However, the product data sheet or COA will indicate when a diluent other than water is required. Recommended solutions, carrier protein concentrations, and extended storage conditions can also be found within these documents.
Reconstitution to a concentration of 0.1 to 1.0 mg/mL is recommended. For example, 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 between 1 mg/mL and 0.1 mg/mL.
In general, we recommend storing lyophilized recombinant proteins at -20 degrees C upon arrival, but short-term storage at 4 degrees C (up to 6 months) or room temperature (up to 30 days) is permissible.
For reconstituted protein solutions, we recommend storing working aliquots (containing at least 10 µL of protein solution with carrier protein) at -20 degrees C to -80 degrees C for extended storage. Do not allow multipe freeze-thaw cycles.
As these storage conditions are protein-dependent, we do recommend checking the product-specific storage recommendations that are provided in either the product manual, data sheet, or certificate of analysis (CoA).
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)).
Carrier proteins help improve the stability of proteins in dilute solutions, extending 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, resulting in significant reduction of protein concentration if the starting concentration was low. 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 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 cases, glycosylation of a growth factor or cytokine does not affect how it binds to a receptor directly, so its biological activity is not significantly affected by glycosylation in in vitro studies. 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 a 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 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 specific activity of a bioactive protein can be determined using the following equation:
1 x 10E6/ED50 (ng/mL) = specific activity (units/mg)
The ED50 can be found on the Certificate of Analysis (COA) for the recombinant protein, but we advise determining the ED50 of a given recombinant protein in your particular functional assay system.
For additional information on ED50, and its relationship with specific activity, please refer to our Tech Tip:https://assets.thermofisher.com/TFS-Assets/BID/Technical-Notes/converting-ed50-ng-ml-specific-activity-units-mg-tech-note.pdf
Where possible, Thermo Fisher Scientific obtains International Unit (IU) values through multiple side-by-side comparisons of our product(s) against the analogous WHO Reference Standard within our biological activity assay. Performing multiple comparison tests allows us to account for any outliers due to possible variations with the assay (e.g., product, handling, assay protocol, etc.) Using the results of these comparisons, we can provide a reliable quantification of our product's activity in relation to the activity of the WHO Reference Standard.
Information pertaining to whether a specific product has been tested against the WHO Reference Standard can typically be located on the product page or Certificate of Analysis (COA).
For cell maintenance: Human recombinant activin A, FGF-basic, and IGF-II (Stem Cells 24:1476 (2006); Nature 448:1015 (2007)).
For human ES cell differentiation: BMP-4, EGF, and HGF (Proc Natl Acad Sci U S A 97:11307 (2000)).
The full length Human FGF-basic (FGF-2/bFGF) (aa 1-155) Recombinant Protein is recommended for stem cells whereas the truncated variant, Human FGF-basic (FGF-2/bFGF) (aa 10-155) Recombinant Protein which is missing the first 9 amino acids, is recommended for use with neural and cardiac cells.
The same growth factors used for ES cell study can also be used for iPS study:
For cell maintenance: Human recombinant activin A, FGF-basic, and IGF-II (Stem Cells 24:1476 (2006); Nature 448:1015 (2007)).
For human ES cell differentiation: BMP-4, EGF, and HGF (Proc Natl Acad Sci U S A 97:11307 (2000)).
No, Leukemia Inhibitory Factor (LIF) Recombinant Mouse Protein, Embryonic Stem Cell-Qualified cannot work in human cells as it does not bind human LIF receptor.
Besides the species difference between LIF Recombinant Mouse Protein, Embyonic Stem Cell-Qualified (Cat. Nos. A35933, A35934, A35935) and LIF Recombinant Human Protein (Cat. No. PHC9484), the other main difference is that ESC-qualified mouse LIF is derived from plant tissue, whereas LIF Recombinant Human Protein is produced in E. coli. ESC-qualified mouse LIF is verified to contain less than 0.005 ng/µg of endotoxin, which is more than ten times lower than that in E. coli-derived recombinant human LIF protein. These two recombinant proteins also have different reconstitution instructions, different storage conditions, and shelf life.
For NSC expansion, the following growth factors are used: recombinant EGF, recombinant FGF-basic, and recombinant VEGF. In addition, several neurotrophins such as BDNF, NT-3, CNTF, and GDNF are also used in the related studies.
For Research Use Only. Not for use in diagnostic procedures.