Help & FAQs: Proteins and Enzymes

• Are R&D Systems recombinant proteins and antibodies sterile?
• Although the vials are bottled using aseptic techniques, heat-treated vials, and sterile stock solutions, they are not considered or guaranteed to be sterile. If sterile material is needed for an experiment, the material can be filtered through a 0.2 micron filter designed for use with biological fluids.
• Can an enzyme be stored after activation?
• The enzyme would remain active for a limited amount of time, but we do not have data to suggest how long the activated enzyme will retain activity. We recommend activating the enzyme and using immediately for each experiment.
• Can R&D Systems recombinant and natural proteins or antibodies be used for in vivo experiments?
• Our proteins and antibodies can be used for in vivo experiments in animals in approved studies, but not in humans or veterinary animals.  R&D Systems does not perform any in vivo testing. Therefore, the activity and half-life in these applications are uncertain. References for in vivo use may be found by clicking on the "Citations" tab on the product specific web page. We recommend using the carrier-free forms of proteins for in vivo experiments and Technical Service can provide lot-specifc endotoxin levels upon request. 
• Can this protein be used in an application other than the one listed on the datasheet?
• The bioassay listed on the protein datasheets describes the method R&D Systems uses to QC the protein. There are many other potential ways to use each protein. We maintain a database of references citing the use of products in other applications or with different cell types. Please click the Citations tab on the product-specific web page or contact Technical Service for more information.
• Do you assign CAS numbers to your recombinant proteins?
• It is our policy not to assign CAS numbers to our recombinant proteins or antibodies unless there is a hazard which needs to be disclosed. While of high purity, they are still considered biological mixtures, and not pure synthesized chemicals.
• Does R&D Systems have biotinylated proteins?
• R&D Systems offers an expanding line of biotinylated proteins, with more information found on the following link: https://www.rndsystems.com/products/active-biotinylated-proteins.  Products can also be easily identified on the Product Search Page (https://www.rndsystems.com/products), and filtering for Conjugate on Biotin.Please inquire if you are unable to find your protein of interest.
• How can I convert your research use only (RUO) grade proteins to units or international units?
• R&D Systems intentionally sells most proteins by mass rather than units. To follow an assay protocol given in units, or to compare a commercially available product sold on a unit basis, one must verify how the unit was defined. It is strongly recommended that a dose response be performed when beginning experiments, or with new lots or vendors. Our recommendation is using the midpoint of our ED50 range as the midpoint in the titration, and also running samples at 5, 10, 20, or 50 fold increments above and below this point. If the author/vendor has compared their "unit" to a WHO standard unit, you may find information contained in the WHO Conversion Table on our website (https://www.rndsystems.com/resources/technical-information/unit-conversion-table) to determine roughly where to start. This table contains approximate mass to unit conversion factors based on our historical testing against WHO standards. Please note this information is for reference purposes only, and is not routinely tested for our RUO grade proteins. The WHO standards are available from the NIBSC for purchase and can be used side-by-side in your own assay to determine specific activity in units.  Alternatively, please also consider our preclinical animal-free (AFL) grade or GMP grade versions which, in addition to ED50, have minimum specific activity specifications and each bottled lot is tested against the WHO standard, with specific activity reported on the lot-specific CoA.
• How do I convert kDa to grams/mol?
• R&D Systems lists the molecular weight of our proteins in kDa on the datasheets. The approximate conversion to molar mass is 1 Dalton = 1 gram/mol. For instance, an 8 kDa protein would be 8000 grams/mol.
• How do I know whether an R&D Systems Recombinant Protein contains a tag (His-tag, GST-tag, Fc domain, etc.)?
• Please refer to the Source section on the product datasheet.  A tag will be explicitly listed if it is included in the expressed protein provided.
• How much BSA is added to R&D Systems proteins bottled with carrier?
• For bottlings with carrier protein, 50 μg of BSA is added per 1 μg of recombinant protein.
• I ordered a vial of 5 μg protein carrier free. Why is there no lyophilized pellet visible in the vial? 
• There are multiple reasons why you may not observe a lyophilized pellet in your vial. The three most common reasons for this observation are:1. The lyophilized product has become dislodged from the bottom of the vial during shipping/handling and is dispersed on the vial wall or cap. Tapping the vial firmly on the bench or a quick spin in a centrifuge may bring the lyophilized product back to the bottom of the vial.2. When our products are bottled with a carrier protein (BSA), this protein is provided as 50 µg of carrier for every 1 µg of product which makes the resulting pellet very pronounced. Conversely, when products are offered carrier-free the pellet can be very hard to visualize due to this lack of carrier.3. Some antibodies, such as the capture antibody in our DuoSet ELISAs, are formulated with an excipient to help protect the antibody during the lyophilization process and improve its long-term stability. Over time, this excipient can absorb moisture and the product appearance may change from a typical lyophilized appearance to a transparent, film like appearance.This is a common observation and we would recommend using the product as normal. Please tap or centrifuge the vial to bring all the material down to the bottom and proceed to reconstitute the product as outlined on the Certificate of Analysis or datasheet. If you wish, you can also test for the concentration of the antibody just after reconstituting using an A280 test (i.e. Nanodrop) or a simple assay such as Bradford.
• I received a lyophilized product at room temperature but the product label and datasheet indicate that it is to be stored at -20 ºC. Is this an error? Will product activity be compromised?
• The product is shipped at ambient temperature by design and will not be compromised. Lyophilization increases product stability, while reducing packing materials and shipping expense. We have performed “stress tests” on our lyophilized products. These products are subjected to 37º C for 3 days in a humidified chamber and found to be stable. These data demonstrate that lyophilized products shipped ambient and received with in a 3 day window retain full activity. We will guarantee and support the product performance when delivered and properly stored within this timeframe. If the product was not delivered within 3 days to your facility or was not stored properly upon receipt, please contact Technical Service.
• I used your recombinant protein as a control in the corresponding ELISA kit. Why am I seeing discrepancy in mass values?
• First, a large dilution is required to place the recombinant protein on the standard curve range. Typically this is a dilution from μg/mL to pg/mL. Any dilution step can introduce inaccuracy and the larger the dilution step the greater the potential for error. Any pipetting error or mis-calibrated pipet can result in apparent over- or under-recovery.Second, R&D Systems immunoassays have been developed to measure a level of protein captured by one antibody and detected by a second antibody. This measurement is calibrated to standards established when the kit was initially developed. The protein determination of these initial standards became the Master Calibrators to which all new standards are formulated. This provides R&D Systems immunoassay kits with consistency between manufacturing lots. In general, we would expect +/- 25% recovery of the amount stated on the vial when using the Quantikine® ELISA to determine a protein concentration. There may be slight differences in the immunologically recognizable mass between lots of protein, so the apparent concentration provided on the vial may vary from lot-to-lot when measured in the ELISA. If you are using proteins to make controls, it is better to value assign the mass based on measurement in ELISA and not use the mass on the vial when setting control levels.
• Is an Fc chimera an antibody?
• No, an Fc chimera is a recombinant protein, not an antibody. A recombinant protein may be fused to the Fc region of IgG for multiple reasons. It can be used for purification, for detecting the protein with an anti-IgG antibody in a binding assay, or to encourage the formation of disulfide-linked homodimers which increases the bioactivity of some recombinant proteins.
• Is carrier free recombinant protein recommended for use as a positive control on a Western blot?
• Yes, we recommend the carrier free protein without the BSA for this purpose.  The presence of 50 μg of BSA to 1 μg of recombinant protein may affect the migration of the recombinant protein in SDS PAGE and may also mask recombinant protein bands on a membrane pre-stained with a protein visualization dye such as Ponceau S. Keep in mind that due to the expression system used and the addition of tags or other modifications to the recombinant protein, it may not migrate at the predicted molecular weight of the native molecule in natural samples.
• Lyophilized proteins have a specific reconstitution condition listed on the product datasheet. Can I dilute the protein prior to storage?
• For optimal stability and performance, we recommend storing the reconstituted protein at the reconstitution concentration in single-use aliquots of a volume no less than 20 microliters. The reconstitution conditions specified for each protein are based on in-house stability testing.  
• The enzyme I received is supplied as liquid.  What would be the best way to store it?
• The enzyme should be stored in individual aliquots, preferably no less than 20 uL, at the original stock concentration. This will provide the best stability for the product.
• The vial is supposed to contain lyophilized protein, but it appears to be empty. Is there anything in it?
• Pellets can be dislodged during shipping and become disbursed on the vial wall and in the cap. Centrifuge or tap the vial on the benchtop to return this material to the vial bottom. If this does not reveal a pellet, closely inspect the cone of the vial. Some pellets appear as only a tiny amount of material or as a transparent film due to the original buffer formulation. This is a normal appearance for many proteins. For example, if the product is originally lyophilized from a solvent such as acetonitrile or ethanol and supplied carrier-free, you may not be able to visualize the pellet with the naked eye. This does not mean the vial is empty. Reconstitute the vial as directed. After reconstitution, protein concentration can be tested with a spectrophotometer.
• The protein I'm trying to order is only available in carrier-free format.  Is it possible to order my protein of interest bottled with carrier protein?
• To investigate whether this is possible, please contact Technical Service (techsupport@bio-techne.com) or Custom Services (https://www.rndsystems.com/services).
• What are the benefits of using Sf-21 cells for protein expression?
• Sf-21 cells are from Spodoptera frugiperda (fall army worm). Sf-21 cells are able to incorporate many post-translational modifications including glycosylation and phosphorylation, although not to the same degree as mammalian cells.  
• What are the glycosylation differences between recombinant proteins produced in E. coli, Sf-21, and NS0 or CHO cells?
• E. coli-derived proteins are not glycosylated. Sf-21 cells are able to partially glycosylate proteins on N-linked sites with 3-9 mannose residues. They do not incorporate galactose or sialic acid, so there is no branching of sugars. Glycosylation on O-linked sites in proteins derived from Sf-21 cells is sporadic. Both NS0 and CHO cells are capable of fully glycosylating N-linked and O-linked glycosylation sites with branching sugars.
• What buffer should I use for dilution of the fluorescent calibration standard used to develop a conversion factor (pmol/RFU) for my enzyme assay?
• The fluorescent standard should be diluted in assay buffer and run in duplicate, reserving wells for assay buffer only to serve as blanks. Wells in the standard curve will only contain the calibration standard diluted in assay buffer. After subtracting the blank RFU, the resulting RFU versus concentration can be plotted to determine RFU/pmol. This slope (RFU/pmol) can then be inverted (pmol/RFU) and used in specific activity calculations.
• What grade BSA is used for the reconstitution of R&D Systems proteins?
• R&D Systems uses a Probumin® Media Grade BSA (Millipore; Catalog # 81-068) for protein reconstitution. It is recommended that customers use the same or equivalent.
• What is R&D Systems doing to reduce its use of packaging from non-renewable resources?
• Environmental stewardship is important to R&D Systems and its employees. R&D Systems is ISO 14001:2015 Certified and as part of this we are continuously reviewing our sustainability practices and "green" options. First and foremost, the energy expended to ship back the styrofoam box is more detrimental to the environment than having the facility re-use or recycle it. Our stance is to encourage our customers to implement a recycling program locally. At R&D Systems we have chosen to reduce the use of styrofoam as much as possible by: doing extensive stability testing in order to determine which products can be shipped with minimal packaging at ambient temperatures, converting the use of non-recyclable packaging materials to recyclable plastics, cardboard, or biodegradable materials, and continuing to investigate alternatives to dry ice shipments, the use of re-usable containers, and gel packs that allow for smaller styrofoam containers. Employees were key to initiating a recycling program at our facilities. Internally, we recycle paper, plastic, cardboard, aluminum, and glass.
• What is the difference between Avi-tag biotinylated proteins and amine- and sugar-labeled biotinylated proteins?
• For sugar- and amine-labeled biotinylated proteins, multiple biotin molecules are conjugated to the protein after purification. The result is a biotinylated protein with a high biotin-to-protein ratio. This make these particular biotinylated proteins good candidates for applications that require high signal, such as flow cytometry, ELISA, SPR, and immunoprecipitation. While sugar and amine biotinylation can produce varying results (as the biotins are conjugated at potentially any free sugar or amine on the protein), Avi-tag biotinylated proteins feature biotinylation at a single site contained within the Avi-tag, a unique 15 amino acid peptide. The DNA sequence coding for the Avi-tag is inserted during cloning and contained in the final expression vector construct. Biotinylation of the Avi-tag is enzymatically performed by the E. coli biotin ligase BirA. Additionally, the consistent labeling enables uniform orientation of the protein when bound to a streptavidin-coated surface. For more information, please see https://www.rndsystems.com/products/active-biotinylated-proteins.
• What is the expiration of R&D Systems proteins and antibodies?
• R&D Systems has established a policy of not limiting the useful life of a product by providing an expiration date or manufacture date for our protein and antibody products. Under proper storage conditions, proteins and antibodies tend to be stable for many years. These conditions include storing proteins as lyophilized powder, storing the product frozen (-20° C or -80° C) at protein concentrations of greater than 0.1 mg/mL, and limiting the number of freeze/thaw cycles. Please see individual product datasheets for specific instructions. Routine quality control testing by our company ensures that all products have acceptable biological activities at the time of sale. R&D Systems can not control storage conditions of a product upon receipt by the end user. In lieu of an expiration date, we choose to offer a warranty on our protein and antibody products. All products supplied by R&D Systems are warranted to meet or exceed our published specification when used under normal conditions in your laboratory.Typically, this warranty will extend 6-12 months from time of purchase. Please see individual datasheets for specific stability claims. If the product fails during the stated period, a replacement product or credit will be issued.  For details regarding our warranty  please see http://www.rndsystems.com/customer_service_legal.aspx.
• What is the Km value of this enzyme?
• We do not conduct Michaelis-Menten studies or determine Km values for our enzymes. Rather, we assay a single substrate concentration in the linear portion of the curve. This enables us to focus on lot-to-lot consistency.
• What is the recommended method for reconstitution of a lyophilized protein or antibody?
• Unless more specific directions are on the Certificate of Analysis provided with the product, we suggest the following procedure to ensure optimal recovery: 1. Allow the vial and reconstitution buffer to equilibrate to room temperature. 2. Briefly centrifuge the vial to ensure that all lyophiliate is collected at the bottom of the vial. 3. Add the amount of buffer required to achieve the concentration recommended on the product insert. 4. Allow the vial to reconstitute for 15-30 minutes at room temperature with gentle agitation, like on a rocker platform or rotating by hand.  Avoid vigorous shaking that can cause foaming and protein denaturation. 5. Aliquot into volumes greater than 20 μL and store as indicated on the product insert. If the vial exhibits flakes or particulates, mix the product for a couple of hours at room temperature and then at 4oC overnight. Please contact Technical Service at techsupport@bio-techne.com if the product does not go into solution.
• What is the recommended reading frequency when reading a 96-well plate in kinetic mode for an enzymatic assay?
• While reading every 30 seconds should theoretically allow for detection of a linear trend, changing the reading frequency to every 10 or 15 seconds may better encapsulate the curve. The reading frequency may need to be optimized for best results, especially if you are using a 384-well plate.
• What volume of buffer is recommended to reconstitute R&D Systems' lyophilized proteins or antibodies?
• The product datasheet and Certificates of Analysis for lyophilized proteins and antibodies will indicate the recommended reconstitution concentration.  Concentration = mass/volume.  Calculate the target volume by dividing mass by concentration after making certain the mass units match.  For example, a 100 μg vial of an antibody may need to be reconstituted at 0.5 mg/mL.  0.5 mg/mL = 500 μg/mL.  100 μg / (500 μg/1 mL) = 0.2 mL, so 0.2 mL (200 μL) of volume would be needed for reconstitution at this concentration.  Alternatively, use the Molarity and Reconstitution Calculators found under the Resources tab on R&D Systems' website.
• What does the /CF suffix seen in some catalog numbers mean?
• CF stands for "carrier free." Typically our recombinant proteins are bottled with 50 μg of BSA (carrier protein) per 1 μg of proteinin order to enhance stability. The carrier-free version does not contain BSA. Usually proteins with BSA can be stored at a more dilute concentration than the carrier-free counterpart, and they may have a longer shelf-life. Generally one will want to purchase the version with BSA when adding the proteins to culture medium or for ELISA standards. Some situations where carrier-free proteins might be used would be in in vivo applications, when labeling proteins, or for other applications where the BSA might interfere with the experiment.
• When reconstituting the recombinant protein I received, do I need to add BSA during reconstitution?
• BSA as a carrier can enhance the stability of a given protein or permit storage at lower concentrations for a great length of time. For certain applications, however, BSA can be problematic. It would not be necessary to add BSA upon reconstitution unless required for your specific application. Please refer to the lot-specific Certificate of Analysis for the recommended reconstitution buffer formulation.
• Which product can be used as a negative control for human proteins which contain an Fc chimera?
• We offer the Recombinant Human IgG1 Fc Protein, CF (Catalog # 110-HG) as a negative control for Fc chimera proteins that use a human IgG1 Fc region.
• Why are some of R&D Systems recombinant proteins offered as an Fc chimera?
• Some R&D Systems proteins are expressed as chimeras fused to the Fc fragment of an IgG antibody. This can offer advantages for certain proteins. For instance, the Fc portion might stabilize the molecule. In addition, biologically active dimeric proteins can be created by using the natural tendency of the Fc portion to dimerize. Receptor-Fc fusions can also result in a recombinant receptor with higher affinity for its ligand compared to the soluble receptor alone. R&D Systems offers the same recombinant Fc portion separately for use as a control (Recombinant Human IgG Fc; Catalog # 110-HG or Recombinant Mouse IgG2A Fc; Catalog # 4460-MG).
• Why do some of R&D Systems proteins require reconstitution in an acidic buffer? Will this buffer hurt the cells?
• Proteins should always be reconstituted and stored with the buffer recommended on the datasheet. Some proteins are extremely hydrophobic at neutral pH. If the suggested buffer is not used to reconstitute these proteins, they do not go fully into solution and much of the material may cling to the sides of the vial. When the protein is used for an assay, it can be diluted to working concentration with media or another suitable buffer which will neutralize the small amount of acid and make it safe to use with live cells.
• Why should I reconstitute the protein in PBS/BSA if it is lyophilized from the same solution?
• The additional BSA in the reconstitution buffer will aid in the recovery and stability of the protein. The volume of PBS in which the product is lyophilized is generally very small. Therefore, PBS reconstitution does not significantly increase the salt concentration when reconstituted according to the datasheet. The presence of additional salt in the stock does not interfere in an assay when diluted to the working concentration. Please contact Technical Service if the volume of PBS prior to lyophilization is a concern. Because products are Quality Control tested in the manner stated on the datasheet, R&D Systems cannot guarantee the performance of products not reconstituted as described.
• Will trehalose affect my conjugation reaction?
• It is possible that the presence of trehalose will interfere in the successful conjugation of a protein. This will depend on the method used, and the customer should investigate this potential prior to purchasing the product.
• Will trehalose affect the performance of the protein or antibody in my specific application?
• We have seen no adverse effect in our bioassays or other approved applications. However, customers are advised to run a control in their assay to determine if the concentration of trehalose in the protein or antibody formulation has any adverse effects.
• How is the isoelectric point (pI) of a protein calculated?
• The isoelectric point is the pH at which a protein has no net electrical charge. R&D Systems calculates pI using amino acid analysis.
• What is trehalose and why is it in the formulation?
• Trehalose is a non-reducing sugar and does not react with amino acids or proteins as part of the Maillard reaction. It is found in nature in many plants and animals. Trehalose is an effective sugar for stabilizing proteins against damage caused by freezing. It can also make the protein more resistant to moisture when lyophilized, resulting in a product that is less likely to precipitate when reconstituted.
• Will trehalose included in the formulation affect the animal if it is injected?
• Trehalose is unlikely to have an effect in vivo. It has been approved as an excipient for use in human injectable drugs.The trehalose used by R&D Systems is derived from Saccharomyces cerevisiae and is determined to be at minimum 98.5% pure by HPAE. 

GMP Recombinant Proteins

• What is the difference between R&D Systems' traditional recombinant proteins and GMP-grade recombinant proteins? 
• Often the sequence, the expression system, and the basic manufacturing SOP for traditional and GMP proteins are the same. This makes the transition from using research-use proteins to GMP as seamless as possible. In addition, compared to traditional proteins, GMP proteins come with extensive documentation for traceability as well as additional quality control testing and quality assurance review.An overview of R&D Systems' GMP-grade Proteins can be found on the web site: https://www.rndsystems.com/products/good-manufacturing-practices-gmp-grade-proteins
• What kind of decrimper do you recommend for use with your GMP protein vials?
• We do not use a decrimper in-house, but we would recommend the end user look for a 13 mm or a 20 mm decrimper device, depending on the vials they receive. Our 1.0 mL retail sized GMP vials would require a 13 mm decrimper; our other vials would require a 20 mm decrimper.