Help & FAQs: Primary Antibodies

  • 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 antibodies reconstituted in PBS be used in an ELISA with Alkaline Phosphatase detection system?
  • After reconstitution with PBS, typically a large dilution (1:1000 or more) in an alkaline phosphate compatible buffer should not cause problems with an alkaline phosphate development system.  End users will need to further optimize and validate for assay conditions.
  • Can cells be stained one day and analyzed for flow cytometry the following day?
  • For optimal results, analyze cells immediately.  If necessary, cells can be fixed in 1-2% paraformaldehyde and analyzed within 24 hours. Background may increase if analyzing is delayed, making low level staining difficult to detect.
  • Can I use your antibodies for a matched pair ELISA with a standard from another company?
  • Yes, but there are potential issues to consider. There can be differences in immunological recognition or the binding between antibody and recombinant protein. This can happen if there is a difference in the folding or sequence of the recombinant protein used as the standard versus the recombinant protein used as the immunogen for the antibody. Protein folding or sequence differences in the antibody-binding region can lead to poor (or no) recognition of the standard.
  • Can R&D Systems recombinant and natural proteins or antibodies be used for in vivo experiments?
  • 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.
  • Does R&D Systems offer sample sizes?  
  • Due to R&D Systems strict manufacturing and QC processes, smaller pack sizes are not currently offered.
  • Does the human Erythropoietin (Epo) polyclonal antibody (Catalog # AB-286-NA) bind Epo if the receptor is not present?
  • AB-286-NA binds Epo if the receptor is not present.
  • Does the human IL-12 polyclonal antibody (Catalog # AB-219-NA) recognize IL-12 p70?
  • In Western blots, this antibody recognizes the p70 heterodimer as well as the p40 and p35 subunits.
  • 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.
  • Is there epitope information for your antibodies?
  • R&D Systems does not determine the epitope for most of its antibodies. Any information we know about the epitope recognized by a particular antibody will be provided in the datasheet.
  • What is R&D Systems doing to reduce its use of packaging from non-renewable resources? R&D Systems should consider re-using styrofoam boxes that are in good condition after delivery.
  • Environmental stewardship is important to R&D Systems and its employees. R&D Systems regularly explores "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 and we can identify a recycling center for styrofoam nearest your facility if necessary. 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, glass, and styrofoam.
  • What is the difference between reducing and non-reducing conditions in Western blot?
  • Reducing conditions in western blot refers to the presence of a reducing agent such as 2-Mercaptoethanol or Dithiothreitol to the sample buffer. This results in the breaking of inter and intra-chain disulfide bonds in the proteins present in the sample, which in turn may lead to change in the migration rate of the reduced proteins.  Non-reducing conditions refers to absence of reducing agents in the sample buffer.
  • What is the expected MW of IgG in SDS-PAGE (reduced and non-reduced)?
  • The molecular weight is approximately 146-150 kDa under non-reducing conditions and under reducing conditions, two bands are observed at 50 kDa (heavy chain) and 25 kDa (light chain).
  • 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 optimal working concentration of the antibody?
  • R&D Systems' product specific pages and datasheets provide a recommended concentration for each validated application that is a starting point for titration. The concentration required will vary from lab to lab due to other variables and thus titration is strongly recommended for optimizing results.
  • 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:Allow the vial and reconstitution buffer to equilibrate to room temperature.Briefly centrifuge the vial to ensure that all lyophiliate is collected at the bottom of the vial.Add the amount of buffer required to achieve the concentration recommended on the product insert.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.Aliquot into volumes greater than 20 uL 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.Contact Technical Service if product does not go into solution.  
  • What is the specificity and cross-reactivity of Catalog # AB-233-NA?
  • For Western blot, less than 5% cross-reactivity was observed with recombinant human (rh) β-ECGF, and no cross-reactivity was observed with rhFGF acidic, FGF-4, FGF-5, FGF-6, FGF-7, and FGF-9.
  • 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.
  • What type of tubes does R&D Systems suggest to use with antibodies in flow cytometry?
  • R&D Systems recommends the use of FALCON polystyrene tubes in flow cytometry.
  • Which ELISA plate is recommended for DuoSet® ELISA Development Kits?
  • R&D Systems offers clear (Catalog # DY990) and black (Catalog # DY991) microtiter plates, and plate sealers (Catalog # DY992). For added convenience, EvenCoat™ goat anti-mouse IgG microplates are available (Catalog # CP001; Catalog # CP002). EvenCoat microplates are clear, pre-blocked, 96-well polystyrene microplates coated with goat-derived antibody specific for the Fc region of mouse IgG. These plates may be used as a solid support for most sandwich ELISAs utilizing a mouse IgG capture antibody and a non-mouse IgG detection antibody. Other applications include competitive ELISA, IgG isotyping, and hybridoma screening/selection. EvenCoat Microplates may also be used with most DuoSet® ELISA Development Kits. Check your DuoSet ELISA datasheet to see if it can be used with EvenCoat Microplates. If there is no indication that it has been tested, please contact our Technical Service Department for more information.  
  • 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.
  • Does the human β-NGF polyclonal antibody (Catalog # AB-256-NA) cross-react with human BDNF, human NT-3, or mouse β-NGF?
  • In Western blot, less than 5% cross-reactivity was observed with recombinant mouse β-NGF and recombinant rat β-NGF, and no cross-reactivity was observed with recombinant human (rh) NT-3 and rhNT-4.
  • It is recommend to dilute the antibody in PBS, however, I intend to use the antibody in an ELISA with Alkaline Phosphatase. What should be used to dilute the antibody?
  • We recommend reconstituting the antibody in PBS and then diluting the antibody to the appropriate working concentraiton in carbonate buffer, pH 8.6. This will dilute the PBS and not cause problems with an alkaline phosphate development system.
  • What epitope does this polyclonal antibody recognize?
  • Polyclonal antibodies generally recognize multiple epitopes because they are generated using the entire immunogen stated on the product datasheet.
  • What is the molar ratio of biotin to antibody in the biotinylated (BAF) products?
  • The biotin:antibody ratio after the labeling reaction is not determined.  R&D Systems uses a 10 molar excess of biotin to antibody in the reaction mixture and estimates there are between 5-14 biotin molecules per IgG.
  • What is the molecular weight of IgG?
  • An IgG protein is comprised of two heavy chains that are approximately 55 kDa each and two light chains that are approximately 25 kDa each for a total molecular weight of approximately 160 kDa.
  • 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 trelahose in the protein or antibody formulation has any adverse effects.

Cell Surface Staining Fluorochrome-labeled Antibodies

Intracellular Staining Fluorochrome-labeled Antibodies

  • Can cells be stained for flow cytometry and analyzed the following day?
  • In most cases, stained cells can be analzyed the following day if they are fixed and stored appropriately. 
  • Can stained cells be analyzed by flow cytometry the following day?
  • Yes.  Complete entire staining protocol and then fix cells with 0.5 mL catalog # FC004 - Flow Cytometry Fixation Buffer (1X) or 4% paraformaldehyde. Wrap the tubes in foil and place at 2-8 °C. Staining intensity may be reduced after fixation and storage overnight.
  • Can this antibody be used on frozen or paraffin-embedded sections?
  • R&D Systems will provide support for any antibody that is validated for the IHC/ICC application in both frozen and paraffin-embedded sections unless otherwise specified on the datasheet. Although we can never be certain an antibody will perform the same way in every type of tissue, we can demonstrate recognition of the fixed antigen. It is our policy that we will should troubleshooting efforts with our technical service department fail to resolve the issue, R&D Systems will offer a product credit toward a future purchase.If the datasheet does not have an IHC/ICC claim, we do not have any in-house or collaborative data available to support this application and we cannot guarantee results. It is up to the user to decide if they wish to use the antibody in their application.Customers may use the Citations tab on the product-specific web page or contact Technical Service to check for references using a product in an application or with a cell type R&D Systems has not tested in-house.
  • Do intracellular flow cytometry antibodies bind to the surface of the cell?
  • It is possible that an intracellular flow cytometry antibody may bind to the cell surface depending on the biological characteristics of the protein, the specific cell type and experimental conditions. It is recommended to include an unfixed cell control to determine if the antibody is staining the cell surface.
  • The fluorescent-conjugated antibody datasheet lists applications other than flow cytometry in the Specificity section. Does this antibody have further utility in those applications?
  • The Specificity section describes the characterization of the unconjugated antibody in applications such as Western blot and Direct ELISA. Cross-reactivity with other species and related molecules is described, as tested.  The fluorescent-conjugated antibody datasheet features flow cytometry data with natural samples.  Please see the Related Products & Information tab for a convenient link to related antibodies evaluated for use in other applications. 

Monoclonal Antibodies

  • What is the rationale for using mannitol as a bulking reagent?
  • Mannitol is a sugar alcohol that improves the consistency of the pellet size during lyophilization. Mannitol also aids in reconstitution and is a commonly used excipient in pharmaceutical protein/antibody products.
  • At what concentration do we use GRO-alpha, GRO-beta, and GRO-gamma for neutralization in bioassay with Catalog # MAB276?
  • Recombinant Human GRO-beta (Catalog # 176-GB) is used at 6 ng/mL, rhGRO-alpha (Catalog # 275-GR) is used at 10 ng/mL, and rhGRO-gamma (Catalog # 277-GG) is used at 20 ng/mL.
  • Can I use your antibodies for a matched pair ELISA with a standard from another company?
  • Yes, but there are potential issues to consider. There can be differences in immunological recognition or the binding between antibody and recombinant protein. This can happen if there is a difference in the folding or sequence of the recombinant protein used as the standard versus the recombinant protein used as the immunogen for the antibody. Protein folding or sequence differences in the antibody-binding region can lead to poor (or no) recognition of the standard.
  • Can this antibody be used on frozen or paraffin-embedded sections?
  • R&D Systems will provide support for any antibody that is validated for the IHC/ICC application in both frozen and paraffin-embedded sections unless otherwise specified on the datasheet. Although we can never be certain an antibody will perform the same way in every type of tissue, we can demonstrate recognition of the fixed antigen. It is our policy that we will should troubleshooting efforts with our technical service department fail to resolve the issue, R&D Systems will offer a product credit toward a future purchase.If the datasheet does not have an IHC/ICC claim, we do not have any in-house or collaborative data available to support this application and we cannot guarantee results. It is up to the user to decide if they wish to use the antibody in their application.Customers may use the Citations tab on the product-specific web page or contact Technical Service to check for references using a product in an application or with a cell type R&D Systems has not tested in-house.
  • Do you epitope map your monoclonal antibodies?
  • Our antibodies are superior for natural protein detection compared to antibodies generated by short synthetic peptides because we use the full length, modified and folded recombinant proteins as immunogens. It would be very labor intensive to epitope map against the full length protein. We find it more effective to generate many clones and empirically screen the clones for performance and specificity in various applications.  Contact your sales rep for information about purchasing clone panels.
  • Does R&D Systems have sequence information for antibodies?
  • R&D Systems does not typically sequence our traditional antibodies purified from serum, ascites, or hybridoma culture supernate.  However, R&D Systems offers recombinant monoclonals with known, defined sequences, but this information is considered proprietary.  R&D Systems offers the option to generate custom recombinant monoclonal antibodies upon request, please visit Antibody Services for more details.
  • Has a product ever been used for an application that is not listed on the datasheet?
  • If a specific application is not listed on the datasheet, it may mean that this product has not been tested in this application, or it may mean that in-house testing in this application did not meet R&D Systems’ specifications. Please check our Citations tab to see if other researchers have published using your application, sample type and/or species. If you would like more information on whether or not an application has been tested, please contact Technical Service at (800) 343-7475.
  • Has the human gp130 monoclonal antibody (Clone 28126; Catalog # MAB228) been tested in Western blot?
  • Our scientists have found that this antibody does not detect linearized, denatured protein.
  • What are R&D Systems matched antibody pairs?
  • R&D Systems matched antibody pairs assist in the development of an ELISA. R&D Systems has demonstrated that the paired antibodies can be used in a sandwich immunoassay to recognize the recombinant protein. We recommend that the investigator have expertise in immunoassay development before attempting to use these products. Each investigator must empirically determine the optimal concentrations for the capture and detection antibodies. The amount of antibodies supplied are not normalized to develop the same number of plates. R&D Systems recombinant cytokines are not calibrated by ELISA and therefore must be mass calibrated before use. The DuoSet ELISA Development Kit product line includes the antibodies, standard, enzyme, and protocol necessary for running an immunoassay, and can often be a better value than purchasing antibody pairs and protein standard.
  • What grade BSA is recommend for use in ELISA and ELISpot Development Systems?
  • The grade of the BSA used has been found to be a critical component for running a successful ELISA. BSA with a minimum purity of 98% is recommended. R&D Systems recommends using the same BSAs used in development of the DuoSet; Catalog # DY995 Reagent Diluent Concentrate 2, Catalog # DY997 Reagent Diluent Concentrate 1. Millipore Probumin® Diagnostic Grade BSA (Millipore, Catalog # 820451) is another example.
  • What is a direct ELISA?
  • In a direct ELISA, a plate is coated with the analyte of interest and a labeled detection antibody is used to verify the presence of the analyte. The direct ELISA may use a colorimetric, chemiluminescent, or fluorescent reporter.
  • What is a sandwich ELISA?
  • A sandwich ELISA uses an immobilized capture antibody specific for the analyte of interest in a sample. After the analyte is bound to the immobilized antibody, a labeled secondary antibody specific for the analyte is used for detection. The analyte is "sandwiched" between the two antibodies. The sandwich ELISA is extremely sensitive, and the values obtained are quantitative when compared with a standard curve.
  • What is the recommended protocol for reconstituting and aliquotting lyophilized proteins and antibodies?
  • Following these guidelines for reconstituting lyophilized material will help ensure complete recovery of the protein or antibody.Tap or briefly centrifuge the vial before opening to dislodge any lyophilized material that may be dispersed on the wall or cap of the vial.Use the buffer and stock concentration recommended in the product datasheet. If you want a stock concentration that is higher than the one recommended, contact Technical Service for specific recommendations.For optimal recovery, reconstitute using room temperature buffer.After adding the buffer, re-cap the vial and invert gently by hand or place on a slow rocking platform. This will allow the reconstitution buffer to coat all the surfaces inside the vial. Do not mix by vortexing or by pipetting the material up and down.Allow the vial to sit at room temperature with gentle agitation for at least 15 minutes before aliquotting or using.Store the reconstituted protein in polypropylene or siliconized tubes. If aliquotting, it is recommended that aliquots be no smaller than 10 µL. In addition, avoid repeated freeze/thaw cycles.
  • 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 ug vial of an antibody may need to be reconstituted at 0.5 mg/mL. 0.5 mg/mL = 500 ug/mL.100 ug / (1 mL/500 ug) = 0.2 mL, so 0.2 mL (200 uL) 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.
  • Why is the Western blot band size observed with a cell or tissue lysate sample different from the reported predicted molecular weight?
  •  The "predicted" molecular weight is based entirely on amino acid sequence (or the size of the protein). However, there are other factors which may play a part in determining the observed size of the protein in a Western blot.Protein modification:  Post-translational cleavage where a larger pro-form of the protein is cleaved into a smaller active form which decreases its size in the gel or post-translational modification where a protein becomes glycosylated (N or O linked sites), phosphorylated or ubiquitinated increasing its size in the gel.The overall net protein charge determined by the amino acid composition may affect the migration speed through the negatively charged SDS of the gel.Trimerization, or dimerization creating multimeric proteins.  The use of  reducing conditions will help to eliminate these interactions.Alternative splicing may produce differently sized proteins from the same gene. 
  • Why might a recombinant protein have a different observed molecular weight in a Western blot compared to a cell or tissue lysate sample?
  • There are a number of reasons why differences may be observed.The recombinant protein may only contain part of the mature amino acid sequence.Post translational modifications may be different in the recombinant protein than in the natural protein. For example, recombinant proteins produced in E. coli will not be glycosylated, which would make them appear smaller on a Western blot than the natural protein if there are glycosylation sites available in the sequence.The recombinant protein may contain a tag (e.g. His, Fc, or a basic or acidic tail). Check the Source section of the recombinant protein datasheet to check for additional tags or sequences that may be present.
  • 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.
  • What epitope does this monoclonal antibody recognize?
  • R&D Systems does not epitope map its antibodies. The antibody is generated using the entire immunogen listed on the datasheet. If a peptide immunogen is used, this will be indicated on the datasheet.
  • What is the degree of crossreactivity of MAB240 when using other forms of TGF-beta?
  • In sandwich ELISA, less than 2% cross-reactivity with recombinant human TGF-b3 and recombinant amphibian TGF-b5.  No cross-reactivity with recombinant porcine or human TGF-b2 in sandwich ELISA.  Detects rhLAP, rhLatent TGF-b1, rhTGF-b2, rhTGF-b3 in Western blot under non-reducing conditions only.
  • What is the difference between AB###, AF###, BAF###, MAB###, and BAM### antibody catalog number designations?
  • AB### designated antibodies are polyclonal antibodies that are purified using Protein A, Protein G or Ion Exchange chromatography. Consequently, the resulting purified antibody is a total IgG fraction and may contain IgG not specific for the analyte of interest. AF### designated antibodies, are antigen affinity-purified and then further purified by ion exchange or size exclusion. Therefore, AF### antibodies are IgG specific only to the antigen. Antibodies that have the designation MAB### are monoclonal antibodies. BAF### and BAM### designated antibodies are the biotinylated versions of the AF### and MAB### designated antibodies, respectively.
  • What is the rationale for using trehalose to stabilize proteins?
  • Trehalose is an effective sugar for stabilizing proteins against damage caused by freezing. It can also make the protein more resistant to moisture gain when lyophilized, resulting in a product that is less likely to precipitate when reconstituted. In addition, it has been used in approved parenteral therapeutics.
  • 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. 

Mouse Lineage Depletion Monoclonal Antibodies

Polyclonal Antibodies

  • What is the rationale for using mannitol as a bulking reagent?
  • Mannitol is a sugar alcohol that improves the consistency of the pellet size during lyophilization. Mannitol also aids in reconstitution and is a commonly used excipient in pharmaceutical protein/antibody products.
  • Can I use your antibodies for a matched pair ELISA with a standard from another company?
  • Yes, but there are potential issues to consider. There can be differences in immunological recognition or the binding between antibody and recombinant protein. This can happen if there is a difference in the folding or sequence of the recombinant protein used as the standard versus the recombinant protein used as the immunogen for the antibody. Protein folding or sequence differences in the antibody-binding region can lead to poor (or no) recognition of the standard.
  • Can this antibody be used on frozen or paraffin-embedded sections?
  • R&D Systems will provide support for any antibody that is validated for the IHC/ICC application in both frozen and paraffin-embedded sections unless otherwise specified on the datasheet. Although we can never be certain an antibody will perform the same way in every type of tissue, we can demonstrate recognition of the fixed antigen. It is our policy that we will should troubleshooting efforts with our technical service department fail to resolve the issue, R&D Systems will offer a product credit toward a future purchase.If the datasheet does not have an IHC/ICC claim, we do not have any in-house or collaborative data available to support this application and we cannot guarantee results. It is up to the user to decide if they wish to use the antibody in their application.Customers may use the Citations tab on the product-specific web page or contact Technical Service to check for references using a product in an application or with a cell type R&D Systems has not tested in-house.
  • Does R&D Systems have sequence information for antibodies?
  • R&D Systems does not typically sequence our traditional antibodies purified from serum, ascites, or hybridoma culture supernate.  However, R&D Systems offers recombinant monoclonals with known, defined sequences, but this information is considered proprietary.  R&D Systems offers the option to generate custom recombinant monoclonal antibodies upon request, please visit Antibody Services for more details.
  • Has a product ever been used for an application that is not listed on the datasheet?
  • If a specific application is not listed on the datasheet, it may mean that this product has not been tested in this application, or it may mean that in-house testing in this application did not meet R&D Systems’ specifications. Please check our Citations tab to see if other researchers have published using your application, sample type and/or species. If you would like more information on whether or not an application has been tested, please contact Technical Service at (800) 343-7475.
  • What are R&D Systems matched antibody pairs?
  • R&D Systems matched antibody pairs assist in the development of an ELISA. R&D Systems has demonstrated that the paired antibodies can be used in a sandwich immunoassay to recognize the recombinant protein. We recommend that the investigator have expertise in immunoassay development before attempting to use these products. Each investigator must empirically determine the optimal concentrations for the capture and detection antibodies. The amount of antibodies supplied are not normalized to develop the same number of plates. R&D Systems recombinant cytokines are not calibrated by ELISA and therefore must be mass calibrated before use. The DuoSet ELISA Development Kit product line includes the antibodies, standard, enzyme, and protocol necessary for running an immunoassay, and can often be a better value than purchasing antibody pairs and protein standard.
  • What grade BSA is recommend for use in ELISA and ELISpot Development Systems?
  • The grade of the BSA used has been found to be a critical component for running a successful ELISA. BSA with a minimum purity of 98% is recommended. R&D Systems recommends using the same BSAs used in development of the DuoSet; Catalog # DY995 Reagent Diluent Concentrate 2, Catalog # DY997 Reagent Diluent Concentrate 1. Millipore Probumin® Diagnostic Grade BSA (Millipore, Catalog # 820451) is another example.
  • What is a direct ELISA?
  • In a direct ELISA, a plate is coated with the analyte of interest and a labeled detection antibody is used to verify the presence of the analyte. The direct ELISA may use a colorimetric, chemiluminescent, or fluorescent reporter.
  • What is a sandwich ELISA?
  • A sandwich ELISA uses an immobilized capture antibody specific for the analyte of interest in a sample. After the analyte is bound to the immobilized antibody, a labeled secondary antibody specific for the analyte is used for detection. The analyte is "sandwiched" between the two antibodies. The sandwich ELISA is extremely sensitive, and the values obtained are quantitative when compared with a standard curve.
  • What is the recommended protocol for reconstituting and aliquotting lyophilized proteins and antibodies?
  • Following these guidelines for reconstituting lyophilized material will help ensure complete recovery of the protein or antibody.Tap or briefly centrifuge the vial before opening to dislodge any lyophilized material that may be dispersed on the wall or cap of the vial.Use the buffer and stock concentration recommended in the product datasheet. If you want a stock concentration that is higher than the one recommended, contact Technical Service for specific recommendations.For optimal recovery, reconstitute using room temperature buffer.After adding the buffer, re-cap the vial and invert gently by hand or place on a slow rocking platform. This will allow the reconstitution buffer to coat all the surfaces inside the vial. Do not mix by vortexing or by pipetting the material up and down.Allow the vial to sit at room temperature with gentle agitation for at least 15 minutes before aliquotting or using.Store the reconstituted protein in polypropylene or siliconized tubes. If aliquotting, it is recommended that aliquots be no smaller than 10 µL. In addition, avoid repeated freeze/thaw cycles.
  • 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 ug vial of an antibody may need to be reconstituted at 0.5 mg/mL. 0.5 mg/mL = 500 ug/mL.100 ug / (1 mL/500 ug) = 0.2 mL, so 0.2 mL (200 uL) 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.
  • Why is the Western blot band size observed with a cell or tissue lysate sample different from the reported predicted molecular weight?
  •  The "predicted" molecular weight is based entirely on amino acid sequence (or the size of the protein). However, there are other factors which may play a part in determining the observed size of the protein in a Western blot.Protein modification:  Post-translational cleavage where a larger pro-form of the protein is cleaved into a smaller active form which decreases its size in the gel or post-translational modification where a protein becomes glycosylated (N or O linked sites), phosphorylated or ubiquitinated increasing its size in the gel.The overall net protein charge determined by the amino acid composition may affect the migration speed through the negatively charged SDS of the gel.Trimerization, or dimerization creating multimeric proteins.  The use of  reducing conditions will help to eliminate these interactions.Alternative splicing may produce differently sized proteins from the same gene. 
  • Why might a recombinant protein have a different observed molecular weight in a Western blot compared to a cell or tissue lysate sample?
  • There are a number of reasons why differences may be observed.The recombinant protein may only contain part of the mature amino acid sequence.Post translational modifications may be different in the recombinant protein than in the natural protein. For example, recombinant proteins produced in E. coli will not be glycosylated, which would make them appear smaller on a Western blot than the natural protein if there are glycosylation sites available in the sequence.The recombinant protein may contain a tag (e.g. His, Fc, or a basic or acidic tail). Check the Source section of the recombinant protein datasheet to check for additional tags or sequences that may be present.
  • 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.
  • What epitope does this polyclonal antibody recognize?
  • Polyclonal antibodies generally recognize multiple epitopes because they are generated using the entire immunogen stated on the product datasheet.
  • What is the difference between AB###, AF###, BAF###, MAB###, and BAM### antibody catalog number designations?
  • AB### designated antibodies are polyclonal antibodies that are purified using Protein A, Protein G or Ion Exchange chromatography. Consequently, the resulting purified antibody is a total IgG fraction and may contain IgG not specific for the analyte of interest. AF### designated antibodies, are antigen affinity-purified and then further purified by ion exchange or size exclusion. Therefore, AF### antibodies are IgG specific only to the antigen. Antibodies that have the designation MAB### are monoclonal antibodies. BAF### and BAM### designated antibodies are the biotinylated versions of the AF### and MAB### designated antibodies, respectively.
  • What is the rationale for using trehalose to stabilize proteins?
  • Trehalose is an effective sugar for stabilizing proteins against damage caused by freezing. It can also make the protein more resistant to moisture gain when lyophilized, resulting in a product that is less likely to precipitate when reconstituted. In addition, it has been used in approved parenteral therapeutics.
  • 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. 

Recombinant Monoclonal Antibodies