Help & FAQs: Primary Antibodies

•  Is it possible to control the orientation of an immobilized antibody? 
• There are published papers that address this topic: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5374010/ https://avs.scitation.org/doi/10.1116/1.4978435 https://link.springer.com/article/10.1007/s13206-019-3103-0
•  Is the use of a golgi blocker recommended prior to intracellular staining for secreted cytokines?
• Generally 3 uM Monesin is used in conjuction with cell stimulation to prevent signal loss.
• 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 R&D Systems antibody which has been validated for Immunocytochemistry (ICC or IF) be used in Immunohistochemistry (IHC)? Can an antibody which has been validated for IHC be used in ICC?
• R&D Systems will support an antibody that has been validated in-house for ICC or IHC in both applications.   Although we cannot guarantee that an antibody will work in all cells or tissues, under all conditions, our validation shows that the antibody recognizes the fixed antigen. We may not have tested the antibody in both ICC and IHC, but R&D Systems will support its use in either application.  
• Can cells be stained for flow cytometry and analyzed the following day?
• In most cases, stained cells can be analyzed the following day if they are fixed and stored appropriately. However, it is recommended that analysis occurs on the same day for best results. 
• Can I measure the concentration of a fluorochrome-conjugated antibody using an A280 reading?
• The A280 reading of a fluorochrome-conjugated antibody is affected by the fluorochrome as well as by the buffer used to prepare the conjugate. A simple A280 /E280   measurement will not accurately reflect the concentration of the conjugated antibody. The concentration can be found on the lot specific Certificate of Analysis (CoA) for all Bio-Techne fluorochrome-conjugated antibodies. 
• Can I store the antibody product at -80 ºC instead of -70 ºC as stated on the datasheet?
• Yes. The antibody product can be stored at -80 ºC instead of -70 ºC. The reason we state -70 ºC as the storage temperature is to accomodate the fact that many -80 º​C freezers do not maintain that temperature uniformly at all times.
• 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?
• 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 sister clones of a given antibody clone belong to a different isotype?
• Yes. Although rare, we have seen examples where sister clones of a given clone belong to a different isotype.
• Do you map the locations of biotinylated residues on your Biotin Antibodies?
• No, we do not map the biotinylated residues.
• Does this Biosimilar antibody have the same sequence as the therapeutic antibody?
• The Biosimilar antibody has the same variable region and constant region sequence as the therapeutic antibody, except for Catalog numbers MAB9677, MAB9575, MAB9577, MAB9589, and their conjugates. These four antibodies have the same variable region as the Biosimilar, but not the same constant region.
• Does trehalose interfere with metal conjugation to antibodies, for CyTOF?
• Trehalose is not reported to interfere with metal conjugation of antibodies, for CyTOF
• For Antigen-Affinity Purified antibodies, does the antibody eluted from an affinity purification column still have any of the affinity ligand associated with it and is there any procedure to separate that ligand from the antibody? 
• There has been no evidence of ligands leaching off the affinity columns used.   The ligand is covalently bound to the resin and remains on the resin when the antibody is eluted.
• How are your antibodies biotinylated?
• All R&D Systems' Biotinylated antibodies are biotinylated via amines. 
• 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.
• If the sample for IHC  is a tissue perfused with formaldehyde fixative solution followed by sucrose and then frozen, what would be the correct method to follow for staining?
• Tissue perfused with formaldehyde fixative solution followed by sucrose and then subsequently frozen should be treated as frozen tissue sample.  IHC staining protocol suitable for frozen tissue should be followed. Here are the links to these protocols: https://www.rndsystems.com/resources/protocols/protocol-preparation-and-chromogenic-ihc-staining-frozen-tissue-sections; https://www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-ihc-staining-frozen-tissue-sections
• In the frozen tissue IHC protocol at  https://www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-ihc-staining-frozen-tissue-sections, how is isopnetane mixed with dry ice?
• In this protocol, isopentane and dry ice are mixed together in a container.
• Is batch-to-batch variability in polyclonal antibody production tested?
• Each lot of polyclonal antibody is tested against a back lot to ensure lot to lot consistency. 
• Is every antibody tested for endotoxin levels? 
• No. Endotoxin level is only routinely measured for antibodies validated for neutralization. To request more information on endotoxin testing of an antibody, please contact techsupport@bio-techne.com. 
• 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.
• The serum from which animal species is recommended as a blocking reagent for IHC and ICC staining?
• 10% Donkey serum works well generally as a blocking reagent. Serum from the same species as the secondary antibody could alternatively be used as a blocking reagent. 
• What does -SP mean with regard to antibody vial size? For antibody catalog numbers ending with -SP on the webpage, the vial label does not include the -SP. What is the explanation?
• SP stands for Small Pack size of antibody. We offer a smaller mass (25 µg) for most antibodies as a way to test the antibody at an affordable price before purchasing the retail size. The antibody vial label indicates the mass of the antibody in micrograms (µg), but not the SP designation. The -SP size should have 25 µg on the vial label.
• What does -SP mean with regard to antibody vial size?
• SP stands for Small Pack size of antibody. We offer a smaller mass (25 µg) for most antibodies as a way to test the antibody at an affordable price before purchasing the retail size.
• 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 Recombinant antibody?
• Recombinant antibodies are antibodies that are generated in vitro through inserting cloned genes into expression vectors. The expression vector is then inserted into a host cell to express the antibody. Our recombinant monoclonal antibody sequences are isolated from a pre-existing hybridoma, or pre-selected rabbit or llama antibody producing clone. By sequencing our antibodies and expressing them recombinantly we can provide an immortal supply with consistent results every time.
• 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 an agonistic antibody?
• An agonistic antibody is an antibody that can bind to a its target on the cell (a receptor) and produce the same activation effects as the cognate ligand. An example antibody is Human CD28 Antibody, Catalog number MAB342.
• 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 amino acid sequence of your antibody?
• The sequence of our antibodies is considered proprietary.  The accession number given on the product specific page is for the immunogen the antibody was raised against.
• What is the difference between fixation for ICC using methanol and acetone versus using 4% PFA? 
• Acetone, ethanol, and methanol are precipitating fixatives. They precipitate proteins at their isoelectric point, whereas formaldehyde is a cross-linking fixative. Mixing both types of fixatives may allow for better fixation of 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 cannot 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 //www.rndsystems.com/customer_service_legal.aspx.
• What is the genus and species of rabbit used to generate recombinant monoclonal antibodies?
• R&D Systems rabbit recombinant monoclonal antibodies are produced from Oryctolagus Cuniculus, commonly referred to as the New Zealand White species.
• What is the mass extinction coefficient (ε280) used for measuring concentration of an antibody using A280?
• For R&D Systems' IgG antibodies, we use a mass extinction coefficient (ε280) of 1.4. This is a predicted and not calculated value.
• What is the molecular weight of IgG antibodies manufactured at R&D Systems?
•  For IgG antibodies, we estimate that the molecular weight is ~ 150 kDa. 
• What is the molecular weight of IgM?
• We do not measure the molecular weight in-house but the molecular weight of IgM is reported to be approximately 900-970 kDa. 
• 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:1. Allow the product to be reconstituted and the reconstitution diluent to reach room temperature.2. Tap or briefly centrifuge the product vial before opening to dislodge any lyophilized material that may be dispersed on the wall or cap of the vial.3. Use the diluent and stock concentration recommended in the product datasheet. If preparing a stock concentration higher than recommended, contact Technical Service.4. After adding the diluent, re-cap the vial and invert gently by hand or place on a slow rocking platform to allow the reconstitution diluent to coat all the surfaces inside the vial.Note: Do not mix by vortexing or by pipetting the material up and down.5. Allow the vial to sit at room temperature with gentle agitation for at least 15 minutes before aliquotting or using.6. Store the reconstituted material in single use aliquots in polypropylene or siliconized tubes. Aliquots of 10 μL or larger are recommended.If the vial exhibits flakes or particulates, mix the product for a couple of hours at room temperature and then at 4 °C overnight. Please contact Technical Service at techsupport@bio-techne.com if the product does not go into solution.
• What is the Research Resource Identification (RRID) number for your antibody?
• We do not register our antibodies with RRID (https://www.rrids.org/).  Researchers may search the RRID database to find matches.
• 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. You can use our online calculator here: www.bio-techne.com/resources/calculators/reconstitution-calculator
• Where on the biotinylated antibody is the biotin conjugated?
• All of our biotinylated antibodies are biotinylated via primary amines.
• 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 the performance of the protein or antibody in my specific application?
• We generally have not seen adverse effects 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.
• 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 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 added to stabilize antibodies and other proteins against damage caused by freezing. It can also make proteins more resistant to moisture when lyophilized, resulting in a product that is less likely to precipitate when reconstituted.
• Will trehalose affect my conjugation reaction?
• Trehalose is not usually expected to interfere with conjugation; we often successfully conjugate antibodies which are bottled with Trehalose using primary amine to NHS ester coupling chemistry. However, for optimal conjugation performance, it is best practice to perform buffer change to remove trehalose using desalting column to a compatible conjugation buffer prior to coupling reaction.
• 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.

Cell Surface Staining Fluorochrome-labeled Antibodies

• Do you have any tips to reduce autofluorescence when staining cells using Flow Cytometry, ICC or IHC?
• There are products in the market available to reduce autofluorescence. Alternatively, the fluorophore can be changed to one that falls in the red or far red region, as there is less autofluorescence in that region. Unstained samples can also be checked to see which channel produces the least autofluorescence. Lasltly, one can use chromogenic detection methods if autofluorescence is difficult to eliminate.
• 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. 

Intracellular Staining Fluorochrome-labeled Antibodies

• 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 have any tips to reduce autofluorescence when staining cells using Flow Cytometry, ICC or IHC?
• There are products in the market available to reduce autofluorescence. Alternatively, the fluorophore can be changed to one that falls in the red or far red region, as there is less autofluorescence in that region. Unstained samples can also be checked to see which channel produces the least autofluorescence. Lasltly, one can use chromogenic detection methods if autofluorescence is difficult to eliminate.
• Is a protein transport inhibtor used in the Flow Cytometry protocol for intracellular staining of secreted cytokines?
• Yes. A protein transport inhibitor is routinely used in the intracellular Flow Cytometry staining protocol of secreted cytokines.  Depending on the cytokine, Monensin works best for some where as Brefeldin A works best for others. 
• 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

• 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 recombinant antibodies converted from monoclonals perform identically to the original monoclonal?
• There may be a slight variation due to the production of the monoclonal antibody in a different system, but the two antibodies generally have similar applications and performance specifications.
• 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.
• 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 chimeric antibody?
• Chimeric antibodies are antibodies that are made up of domains from different species. For example, the Fc region or all the constant region domains of a mouse monoclonal antibody may be replaced with constant region domains of a human antibody.
• What is the purity of a monoclonal antibody produced at R&D Systems?
• We purify the hybridoma culture supernatant via Protein A or Protein G chromatography, which is specific for IgG binding. Based on our extensive experience and testing strategies, we expect the purity of our monoclonal antibody products to be very high. An SEC analysis is run for each new lot of antibody. The high purity of our monoclonal antibodies makes them suitable for conjugation to a variety of molecules. 
• 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. You can use our online calculator here: www.bio-techne.com/resources/calculators/reconstitution-calculator
• 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.
• 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.
• 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 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.

Polyclonal Antibodies

• 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.
• For Antigen-Affinity Purified antibodies, does the antibody eluted from an affinity purification column still have any of the affinity ligand associated with it and is there any procedure to separate that ligand from the antibody? 
• There has been no evidence of ligands leaching off the affinity columns used.   The ligand is covalently bound to the resin and remains on the resin when the antibody is eluted.
• 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 chimeric antibody?
• Chimeric antibodies are antibodies that are made up of domains from different species. For example, the Fc region or all the constant region domains of a mouse monoclonal antibody may be replaced with constant region domains of a human antibody.
• What is the purity of an affinity-purified polyclonal antibody produced at R&D Systems?
• We purify polyclonal antibodies using an antigen-affinity column and we expect the purity of our polyclonal antibody product to be very high. A SEC analysis is run for each new lot of antibody. The high purity of our polyclonal antibodies makes them suitable for conjugation to a variety of molecules. 
• 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 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. You can use our online calculator here: www.bio-techne.com/resources/calculators/reconstitution-calculator
• 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.
• What epitope does this polyclonal antibody recognize?
• Polyclonal antibodies generally recognize multiple epitopes.
• 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.

Recombinant Monoclonal Antibodies

• Does this Biosimilar antibody have the same sequence as the therapeutic antibody?
• The Biosimilar antibody has the same variable region and constant region sequence as the therapeutic antibody, except for Catalog numbers MAB9677, MAB9575, MAB9577, MAB9589, and their conjugates. These four antibodies have the same variable region as the Biosimilar, but not the same constant region.
• What is a chimeric antibody?
• Chimeric antibodies are antibodies that are made up of domains from different species. For example, the Fc region or all the constant region domains of a mouse monoclonal antibody may be replaced with constant region domains of a human antibody.
• Why is Western blot listed as an application on a hybridoma version of a monoclonal antibody, but not for the corresponding recombinant conversion of that antibody?
• We choose to not add a Western blot claim on the recombinant conversion of a monoclonal antibody if the only sample tested was a recombinant protein and not a lysate.