General questions

Recombinant Protein Information and Handling

  • 1.  What types of recombinant proteins do you offer? In what format are they provided?

    The majority of the recombinant proteins GenScript offers are cytokines, growth factors and chemokines. We are continually expanding our list of recombinant proteins to include Neurotrophins, virus antigens, and enzymes as well. GenScript provides recombinant proteins as sterile, lyophilized, carrier-free aliquots with a mild buffer. There are no additional proteins or preservatives added to our recombinant proteins.

  • 2.  How are your recombinant proteins produced? What are the advantages of eukaryotic expression (yeast, insect and mammalian cells) exist versus expression from E. coli?

    Most of our recombinant proteins are produced in eukaryotic systems, but the certificate of analysis will provide you with the specific information on how individual products were produced.
    Eukaryotes have several advantages as an expression system.

    • Eukaryotes inherently secrete very few native proteins, but they do secrete recombinant proteins. Therefore secretion is typically used as the first step in the purification process from a eukaryotic system. Recombinant proteins made in E. coli are often localized to inclusion bodies. Purification from inclusion bodies can require harsh conditions to free the recombinant protein, followed by subsequent refolding processes. These harsh treatments can negatively affect the function of the protein.
    • Recombinant proteins secreted by eukaryotes are processed by the Golgi apparatus, and thus they can be post-translationally modified. These modifications include glycosylation, phosphorylation and sulfation. There are many proteins that require modifications for protein function, proper folding or solubility.
    • Eukaryotes do not have a bacterial cell wall like E. coli, thus there is no endotoxin (lipopolysaccharide) present that can affect inflammatory responses in your target system.
  • 3.  When I opened the vial, I didn’t see anything. How do I know there is protein in the vial?

    Centrifuge the vial prior to opening! Most of our products are lyophilized with a low concentration buffer, so the few micrograms of product may not be very visible. We recommend centrifuging the vial in a micro-centrifuge for 20-30 seconds before opening to drive any protein that may be lodged in the cap or on the side of the tube to the bottom of the vial. Our quality control procedures assure that each vial contains the correct amount of product.

  • 4.  How do you reconstitute lyophilized powder? What is an acceptable volume for recombinant protein reconstitution? What concentration is the recombinant protein upon reconstitution?

    Please check the certificate of analysis included with your shipment for the exact instructions for reconstitution because not all products are reconstituted with the same conditions. In general, we recommend using sterile water for reconstitution. Add the recommended volume of sterile water to the vial, and gently shake it to solubilize the protein completely. Do not vortex.

    Typically, 100 µL is an acceptable reconstitution volume for our 10 µg and 50 µg vials of recombinant protein. Our 10 µg and 50 µg vials have a maximum volume of 500 µL. Typically, 1 mL is an acceptable reconstitution volume for our 100 µg vials of recombinant protein. Our 100 µg vials have a maximum volume of 2 mL.

    The concentration of recombinant protein is dependent on the reconstitution volume. For example, if reconstituting 10 µg of recombinant protein in 100 µL carrier protein solution, the concentration is 10 µg/100 µL or 100 µg/mL.

  • 5.  How should I store recombinant proteins? What is the shelf life of your recombinant proteins?

    For longer term storage the protein solution should be stored with a carrier protein (e.g. 0.1% BSA or 0.1% HSA) in working aliquots and stored frozen at -20°C. Please keep in mind that every freeze/thaw cycle may cause some denaturation of the protein. The majority of recombinant proteins have a guaranteed shelf life of one year, unless indicated otherwise on the certificate of analysis. This guarantee is provided if they are kept under optimal storage conditions as stated on the certificate of analysis.

  • 6.  What is a carrier protein?

    Carrier proteins such as HSA or BSA are used to improve the stability of the reconstituted proteins, and help to avoid the product sticking to the walls of the vial.

  • 7.  How do you determine the quantity of your recombinant proteins? Why is the quantity of protein generated by my assay different from your results?

    We determine the quantity of recombinant proteins by BCA, SDS-PAGE, HPLC and other methods. Different assays generate different quantification results. Sometimes the discrepancy can be significant if you conduct a different assay. It is also possible for proteins to form aggregates during storage which cause loss after reconstitution and centrifugation. We run quality control tests for each batch of product, however, it is still possible that a few vials may not be the same as others within the same batch (this rarely occurs).

  • 8.  Why are some proteins fused to tags? Do protein tags affect protein activity?

    Most GenScript recombinant proteins are tag-free. Protein tags however, are useful for several different purposes.

    • Protein tags are useful for protein purification.
    • Tags are used for protein detection in Western blot or ELISA when the specific antibodies are not available.
    • The Fc tag stabilizes molecules, which may increase the half-life of the linked products. Since the Fc fragment tends to dimerize, it helps the linked protein, particularly receptors, to form biologically active dimers.

Protein tags may or may not affect the protein’s activity. For some applications, small tags, such as the His-tag, may not affect protein activity and do not need to be removed. For example, there are more than 100 structures of His-tagged proteins in the Protein Data Bank. This indicates that the small His-tag often does not interfere with correct protein folding. Additionally, tested activity results are listed on our protein web pages. If you have concerns about tags interfering with protein activity and there is no activity data online, please feel free to contact us for latest information at .

Specific Activity & Experimental Design

  • 1.  What is the specific activity of your recombinant proteins? What is meant by a “unit” of protein activity?

    The biological activity (ED50) (or “unit”) for each recombinant protein is available on the certificate of analysis. The ED50 is defined as the protein concentration at which the activity is 50% of the maximum response and is reported in ng/ml. This method of expressing activity should only be used for proteins whose dose-response curves are sigmoidal in shape. The formula for converting the activity as an ED50 to specific activity is:

    Please note that GenScript does not use the International Standard provided by WHO (National Institute for Biological Standards and control) for measuring recombinant protein activity. There is not a way to convert between these “International Units” and the ED50. The best way to compare the activity of recombinant proteins from different sources is to do the same bioassay side-by-side using the same system.

  • 2.  How does the activity of your recombinant proteins compare to competitors’? Do you test the bioactivity of your recombinant proteins with in vivo assays?

    We quality control each and every lot of recombinant proteins. Not only do we check its bioactivity, but we also compare it against other commercially available recombinant proteins. We make sure each recombinant protein’s activity is at least as good as or better than the competition’s. In order to provide you with the best possible product, we ensure that our testing process is rigorous and thorough. We typically validate the activity of the proteins with in vitro assays as described on the technical datasheet and not with in vivo testing.

  • 3.  Does the specific activity of a recombinant protein vary between lots?

    Specific activity is unique for each lot and for the type of experiment that is done to validate it. We recommend that you perform your own specific experimental validation to find out the optimal ED50 for your system.

  • 4.  I want to try to do an experiment with your protein, but the bioassay you use for determining activity is not the same as my application. Will my application work with your protein?

    GenScript products are used for many different purposes, so it would be impossible to predict every possible application. Our standard bioassay is used to confirm an accepted activity level for the product. Our proteins can be used at a broad concentration range, in many different applications, thus, it is the end user’s responsibility to determine the concentrations that work best for their specific assays.

  • 5.  Can I use recombinant proteins from different companies for my ELISA?

    You could use recombinant proteins from different companies for your ELISAs, however keep the following in mind:

    • Protein tags are useful for protein purification.
    • Recombinant proteins expressed from E.coli from the same source can show greater than 10 fold difference in terms of immunoreactivity from lot to lot, primarily due to refolding inconsistency.
    • Different kit standards can be produced and calibrated against different references. So far there is no universally accepted standardization for recombinant protein immunoreactivities.
    • Any changes to the reagents (standards, antibodies, matching matrices) and protocols may affect the final assay performance.
  • 6.  Do most proteins show cross-species activity?

    Species cross-reactivity must be investigated individually for each product. Many human cytokines will produce a nice response in mouse cell lines, and many mouse proteins will show activity on human cells. Other proteins may have a lower specific activity when used in the opposite species.



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