How is the purity for Gibco recombinant proteins and Gibco PeproTech recombinant proteins determined?
Protein purity is most often determined by SDS-PAGE; sometimes, it is analyzed by HPLC as well. Please refer to either the product manual, data sheet or certificate of analysis (CoA) for product-specific purity levels and methods.
Are Gibco recombinant proteins and Gibco PeproTech recombinant protein cross-reactive with other species?
Cross-reactivity depends on the protein and species. In general, human proteins are often active on mouse cells. Many mouse proteins are active on human cells, but they may show a different specific activity than the human protein.
What is the ED50 of my Gibco recombinant protein or Gibco PeproTech recombinant protein?
The ED50 value is defined as the protein concentration that shows a 50% maximal response.
ED50 values are provided as a range because of variability in the bioassay used to determine ED50 values. For calculations using ED50, we recommend using the midpoint of the range.
For additional information on ED50, 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
Can I filter sterilize cell culture medium containing Gibco recombinant proteins or Gibco PeproTech recombinant proteins?
Yes, cell culture medium containing low concentrations of proteins can be filter-sterilized if needed. We recommend the use of a low-protein-binding 0.22 micron filter.
How do I convert the ED50 of a protein (ng/mL) to specific activity (units/mg)?
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 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
How should I store Gibco recombinant proteins and Gibco PeproTech recombinant proteins?
In general, we recommend storing lyophilized recombinant proteins at -20 degrees C upon arrival. However, short-term storage at 4 degrees C (up to 6 months) or at 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).
Some of the Gibco recombinant proteins are not biologically active. Can these be used for cell culture?
No, cell culture usage requires biologically active proteins. Recombinant proteins with known biological activity are indicated by the application “Functional Assay (FN)” on the product web page and data sheet. Whether a particular protein has been tested for bioactivity can be found on its product page. To find proteins with the FN designation, filter by Application on the recombinant proteins search page: https://www.thermofisher.com/antibody/protein/filter/type/Recombinant
What concentration of bovine serum albumin (BSA) do I need to use in dilute solutions for Gibco recombinant proteins and Gibco PeproTech recombinant proteins? Why is BSA required when making a dilute solution?
Generally, a 0.1% solution of BSA is used when making dilute solutions of Gibco recombinant proteins and Gibco PeproTech recombinant proteins, but sometimes, as as high as 1% BSA is used. Protein solutions should be stored with carrier proteins (e.g., BSA) for extended storage, as it improves protein stability.
What are the growth factors and cytokines used for induced pluripotent stem (iPS) cell study?
What kinds of growth factors are used in human embryonic stem (ES) cell research?
What is the relationship between specific activity units and International Units of activity?
There is no direct correlation or calculation between specific activity unit and International Unit (IU) values. International Units (IU) express a quantification of activity for the base amount of a substance in relation to an analogous reference standard with an internationally accepted unit of biological potency (i.e., IU/ng) that has been assigned based on an International Collaborative Study conducted by the World Health Organization (WHO). WHO Reference Standards are made available by the National Institute for Biological Standards and Control (NIBSC). Intended to simplify the comparison of activity of a substance obtained from different sources, IU measurements can vary as comparison methods are rarely the same between sources. A true direct comparison requires standardized methods of analysis in order to guarantee comparability of the substance’s activity in relation to its mass across sources.
Why can't I see the protein pellet in the vial for my lyophilized recombinant protein? Is this normal?
Gibco recombinant proteins are frequently formulated without carrier proteins or additives (e.g., BSA, HSA, sucrose, etc.) and Gibco PeproTech recombinant proteins don't contain a carrier protein. As a result, during lyophilization, the protein product may be deposited on the vial as a thin, and sometimes invisible film instead of a pellet. The size of the pellet, if any, is not directly related to the quantity of the recombinant protein in the vial. Our quality control procedures assure that each vial contains the correct amount of product.
To ensure complete recovery of protein product, before opening a vial of lyophilized recombinant protein, we recommend centrifuging it in a microcentrifuge for 20-30 seconds to drive any protein that may be lodged in the cap or on the side or the bottom of the vial. After reconstitution, you can confirm the presence of product protein by running a small amount on SDS-PAGE. In general, a protein band with expected size should be visible with as little as 10 ng of protein loaded on an acrylamide gel.
Does a particular recombinant protein contain the wild-type or mutated amino acid sequence?
Protein sequence information is available on the individual product page, except for proprietary engineered proteins.
What kind of tubes should I use for storing aliquots of reconstituted recombinant proteins?
In general, we recommend using polypropylene tubes for storing aliquots of reconstituted recombinant proteins. Specific information for appropriate storage and handling of many recombinant proteins can be found on the product pages.
Does each lot of recombinant protein have the same specific activity?
No, each lot can vary slightly with regards to its specific activity. The information on the data sheet for each product is provided as a guide. For lot-specific information, please please check the Certificate of Analysis or contact Technical Support.
What is the specific activity of my recombinant protein?
The ED50 for each functionally active recombinant protein can be found on its product page under Activity as well as in the data sheet. The specific activity (units/mg) for each recombinant protein can be determined from the ED50 using information available in our Tech Tip here: https://assets.thermofisher.com/TFS-Assets/BID/Technical-Notes/converting-ed50-ng-ml-specific-activity-units-mg-tech-note.pdf
What is the shelf life of your recombinant proteins?
The majority of our recombinant proteins have a guaranteed shelf life of one year unless otherwise indicated in our technical data sheets. This guarantee applies only if the recombinant protein is stored under the conditions stated in the data sheet. If you are not completely satisfied with the performance of the product, please contact Technical Support at techsupport@thermofisher.com for assistance.
It looks like the recombinant protein vial I received from you is empty. Is this normal?
Gibco recombinant proteins are frequently formulated without carrier proteins or additives (e.g., BSA, HSA, sucrose, etc.) and no Gibco PeproTech recombinant protein contains a carrier protein. As a result, during lyophilization, the protein product may be deposited on the vial as a thin, and sometimes invisible, film instead of a pellet. The size of the pellet, if any, is not directly related to the quantity of the recombinant protein in the vial. Our quality control procedures assure that each vial contains the correct amount of product.
To ensure complete recovery of protein product, before opening a vial of lyophilized recombinant protein, we recommend centrifuging it in a microcentrifuge for 20-30 seconds to drive any protein that may be lodged in the cap or on the side to the bottom of the vial. After reconstitution, you can confirm the presence of product protein by running a small amount on SDS-PAGE. In general, a protein band with expected size should be visible with as little as 10 ng of protein loaded on an acrylamide gel.
Why does my recombinant protein not show activity in my own experiment?
Assay time is critical. Each assay needs to beoptimized and performed at the peak response time. Different cells may respond differently to a growth factor or cytokine. We suggest repeating our QC assay using same indicator cells as suggested in the manual to see if you can obtain a similar response. In addition, serum may be masking the response. Serum starvation might be needed for certain types of assays.
Why does my recombinant protein not have the activity claimed by the product materials?
If you carried out a test as described in our product insert and did not see any response, this could be due to several possibilities listed below:
- Protein was not reconstituted according to the instructions.
- The reconstituted protein is too old or protein might have precipitated. We recommend using the reconstituted protein within 3-6 months after reconstitution.
- Carrier protein was not added for proteins reconstituted at a concentration <0.1 mg/mL. Working solutions at <0.1 mg/mL should be used immediately; we do not recommend long-term storage of solutions at this concentration.
- The protein solution was exposed to multiple freeze/thaw cycles or was exposed to high temperature.
- Proteins were handled in the wrong types of vessels (some proteins are very sticky to certain plastics).
Why is my recombinant protein not soluble?
The solubility issue might be due to improper handling, or use of a solvent other than the one we recommended. We recommend that you warm the lyophilized powder to room temperature before you open the vial, and that you solubilize the protein in the buffer solution recommended in the manual (some proteins are more soluble in low pH buffer). Do not reconstitute at a protein concentration greater than 1 mg/mL. Do not vortex or mix protein solutions vigorously. Allowing the reconstituted protein to incubate overnight at 4 degrees C may help resolve any solubility issues.
Are Thermo Fisher Scientific recombinant proteins calibrated with the WHO Reference Standard?
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 COA.
How is the biological activity of a recombinant protein determined?
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.
Does glycosylation affect the biological activity of recombinant proteins?
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.
What are the major differences between the recombinant proteins produced in different expression systems?
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.
Why do I have to freeze recombinant protein solutions in working aliquots?
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)).
How should I store the reconstituted recombinant proteins?
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
How should I reconstitute Gibco recombinant proteins and Gibco PeproTech recombinant proteins?
Depending on 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.
Why are carrier proteins needed in some of the recombinant protein solutions?
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.
How do I store my lyophilized recombinant protein?
Lyophilized proteins can typically be stored at 2 to 8 degrees C for several weeks, or stored dessicated at -20 degrees C for long-term storage.
