Frequently Asked Questions

GENERAL

How are Symansis' products different to those available from other suppliers?
Will antibodies raised against E. coli expressed cytokines recognize the Symansis human expressed cytokine?
What are Fc fusion proteins and why are they used?
How are the 2D densitometry images made?

RECONSTITUTION & USE

What re-suspension buffer should I use?
What are the recommended storage conditions for maintenance of optimal longer-term biological activity?
Are all products supplied sterile?
Does the product contain carrier or any other additives?

SUGAR STRUCTURE

What is the difference between the theoretical molecular weight and the observed molecular weight?
What is the difference between the theoretical pI and the observed pI?
Why does the 2D gel show multiple spots for my protein?
Why does the protein MW change after treatment with PNGase F?
How do you know the protein has N-linked glycans?
What is the glycosidase cocktail used?
Why is the observed molecular weight a range not a precise value?
Why is the 1D gel band broad?
Why is a glycosidase cocktail used?
How do you know the protein has O-linked glycans?

ELISA

What is the advantage of Symansis' human cell expressed ELISA standards?
Do I have to run all of my standards and samples in duplicate?
Do I have to run all of my samples at one time?
What type of reproducible results are obtained with the assays?
What storage conditions are needed for a kit or product?
Is it possible to store the reagents other than indicated?
How should I store my samples?
How many samples will each kit run?
Can I modify the protocol?
Do I have to run a Blank, or Zero Standard every time?
Can I alter the volume of sample I use in the assay?
What should I do if the reagent volume is not enough?
Can components from different kits be used?
Can I store the diluted standards and use them again?
Do I make up my own standard curve?
How do you recommend I wash my plate?
Do I need to use a plate shaker?
Can I use an orbital flask shaker instead of a plate shaker?
Why do I have to use wavelength correction between 450-570nm?
If I extract my sample, do I still need to follow the recommended dilutions given in the kit insert?
What is the expected concentration of analyte that I should expect to find?
My optical densities were a little higher (or lower) than those in the Package Insert that came with my kit. Why?
Can Bovine Serum Albumin (BSA) be replaced by other things such as Fetal Bovine Serum (FBS), in the ELISA buffers?
My samples are in tissue culture medium; do I need to dilute my standards in the same medium?
For how long can I store my capture antibody-coated plates?
What are the reasons for High Background?
What are the reasons for Incorrect Standard Curves?
What are the reasons for No Color Development?
What are the reasons for High Intra Assay Coefficient of Variation (CV) (data variation)?
When is it more appropriate to use a Chemiluminescent Immunoassay instead of an ELISA?
What if I am not obtaining good results or am having difficulties?
Why are Symansis products more sensitive standards in quantitative immunoassays involving native human proteins?
 

GENERAL

How are Symansis' products different to those available from other suppliers?
Symansis' products are expressed from human cells, rather than from E. coli, CHO or insect cells. Expression from human cells facilitates post-translational modifications (PTMs), such as phosphorylation and glycosylation of the peptide backbone. PTMs may affect protein stability, protein-protein interactions, protein folding, and antigenicity. Non-human expression systems do not add certain PTMs and may add other PTMs incorrectly (e.g. Insect cells are known to overglycosylate exogenously expressed proteins). Expressing human cytokines from human cells ensures that the proteins will more closely mimic the naturally occurring cytokines. For further information on this area please visit our Resource Centre pages. 

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Will antibodies raised against E. coli expressed cytokines recognise the Symansis human expressed cytokine?
Symansis' cytokines are different from our competitor's because the cytokines are expressed from human cells. This means that the cytokines have human post-translational modifications, such as glycosylation. It is possible that the antibody-binding sites may be masked by glycoproteins. Glycosylation may also facilitate protein folding into a more stable state, internalising antibody-binding sites (for antibodies derived against E. coli expressed cytokines). recombinant expression of human cytokines from NSO, CHO or insect cells (e.g. Sf21 cells) also facilitates glycosylation, but these expression systems may add glycan structures that are different when compared with the naturally occurring cytokine. It is anticipated that Symansis' human expressed cytokines will behave differently to cytokines expressed using different expression systems such as E. coli or CHO. The exact difference observed would depend upon whether the antibody is monoclonal or polyclonal, and the epitope that the antibody recognizes. Regardless, it is believed that the behavior of Symansis cytokines more closely mimics naturally occurring human cytokines.

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What are Fc fusion proteins and why are they used?
Cytokines and particularly the extra cellular domains of cytokine receptors may be expressed as Fc fusion proteins. The Fc region comprises the hinge region and the CH2 and CH3 domains of human IgG1. The Fc domain facilitates the dimerisation of the fusion protein via the formation of disulfide bonds. This in turn will greatly increase the neutralising ability of the protein relative to monomeric receptor forms. Additionally, Fc fusion proteins are known to exhibit increased half-life in vivo. Expression of an Fc fusion protein can increase the binding affinity of particular receptors relative to that of the soluble monomer. This provides the receptor-Fc dimer a number of advantages over the corresponding monomeric receptor equivalents (e.g. soluble Interleukin receptor monomers). Advantages include increased protein half-life, increased ligand binding affinity, and minimising the natural membrane bound receptor complexes.

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How are the 2D densitometry images made?
The Symansis product was focused on a pH 3-10 gradient IPG strip and then run on a linear gradient SDS-PAGE gel along with and a set of molecular weight markers. The gel was stained using Deep Purple or Coomassie Blue protein stains. The relative intensities of the proteins spots visible on the 2D-gel were analysed using the software ImageJ software(http://rsb.info.nih.gov/ij/). Densitometric analysis was performed on the spots within a selected area of the gel and a background subtraction was conducted using the appropriate blank region of the gel (region lacking protein spots). Volume integration was performed on each protein spot of interest, and from this the centre of mass and its relative percentage intensity were calculated. The molecular weights and pI values of the respective spots were determined by comparing them to the position of the precision MW markers and the linear IGP strip. The former were fitted to an exponential function with a 4th order polynominal to interpolate protein spot locations accurately between MW marker locations. Each protein spot corresponds to a unique isoform of the Symansis product.

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RECONSTITUTION & USE

What re-suspension buffer should I use?
We recommend that each lyophilised cytokine be resuspended in 500ul 1 x sterile PBS. If the cytokine is to be stored for a long period of time, we recommend a concentration of no less than 100ug/ml.

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What are the recommended storage conditions for maintenance of optimal longer-term biological activity?
Our products are lyophilised to provide stability over a range of temperatures. However we recommend long-term storage of lyophilised products at -20oC. When cytokines have been resuspended, we recommend short-term storage at 2-8o C. For longer-term storage we recommend aliquotting the solution into single use vials (to avoid repeated freeze-thaw cycles) and storing the vials at -20o C, or -80o C

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Are all products supplied sterile?
All products are supplied sterile and suitable for use in tissue culture or other sterile environments.

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Does the product contain carrier or any other additives?
Symansis' cytokines are formulated with carrier proteins to enhance the stability of the product. We also carrier-free cytokines on request. Should you be interested in purchasing carrier-free cytokines, please email info@Symansis.com outlining which cytokine/s you are interested in purchasing carrier free and in what quantity. We will liaise with you to deliver the product to you as soon as possible.

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SUGAR STRUCTURE

What is the difference between the theoretical molecular weight and the observed molecular weight?
The theoretical molecular weight is calculated based on the amino acid sequence of the mature protein. The theoretical molecular weight accounts only for the protein backbone after removal of the signal peptide and any pro-peptides, it does not include any post-translational modifications (PTMs) that may have occurred during the production of the protein. Most PTMs don't contribute significantly to the molecular weight (MW) of the overall protein. For example, a phosphate group has a MW of 80 Da and acetylation adds 40 Da. The exceptions to this are glycosylation, lipidation, and the addition of ubiquitin. Since all products from Symansis are soluble, lipidation and ubiquitin addition can be ignored. Symansis proteins are expressed out of human cells, facilitating the addition of oligosaccharides (glycans) to the protein backbone. The attachment of glycans to the protein backbone increases the observed molecular weight above the theoretical molecular weight. Most N-linked glycan structures have a MW greater than 2 kDa. If there are 3 occupied N-glycan sites on the protein, the MW of the protein can be increased by more than 6-10 kDa. Thus the difference between the theoretical molecular weight and the observed molecular weight may be used to derive the estimated percentage glycosylation of the cytokine. 

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What is the difference between the theoretical pI and the observed pI?
The theoretical pI is normally calculated from the amino acid sequence of the mature protein. The observed pI differs due to post-translational modifications (PTMs) including phosphorylation and sialylation. For example, the theoretical pI of IL-10 is approximately 7.9. When a phosphate group is added to a serine or threonine residue, the theoretical pI changes to 7.1. *pI's calculated using ProMOST (http://proteomics.mcw.edu/promost/)

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Why does the 2D gel show multiple spots for my protein?
Multiple spots are due to different protein isoforms arising from post-translational modifications (PTMs) and/or proteolytic processing. It is not due to protein impurities.

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Why does the protein MW change after treatment with PNGase F?
N-linked glycans are removed by PNGase F treatment. A drop in MW shows that the protein is N-glycosylated.

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How do you know the protein has N-linked glycans?
A drop in MW after treatment with PNGase F signifies the presence of N-linked glycans.

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What is the glycosidase cocktail used?
The glycosidase cocktail used to remove N-linked and O-linked glycans consists of PNGase F, sialidase A (neuraminidase), O-glycanase, beta-1,4-galactosidase, and beta-N-acetylglucosaminidase. The sample was incubated in the enzyme cocktail at 370C for 3 hours. 

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Why is the observed molecular weight a range not a precise value?
Proteins with post-translational modifications, in particular glycosylation, appear as broad bands on SDS-PAGE. The observed molecular weight stated is measured from the SDS-PAGE gel calibrated against molecular weight standards.

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Why is the 1D gel band broad?
Proteins with post-translational modifications, in particular glycosylation, appear as broad bands on SDS-PAGE due to the presence of different isoforms and glycoforms.

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Why is a glycosidase cocktail used?
There is no enzyme available to remove all O-linked glycans. The enzyme O-glycanase only removes simple O-linked glycans. The addition of other glycosidases to the mixture ensures that any elongated O-linked glycans are trimmed back to simple structures that O-glycanase can remove.

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How do you know the protein has O-linked glycans?
A drop in MW after treatment with the glycosidase cocktail signifies the presence of N-linked and/or O-linked glycans. If the observed MW of the protein treated with the glycosidase cocktail is lower than the observed MW of the protein treated with PNGase F, then the protein is O-glycosylated.

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ELISA

What is the advantage of Symansis' human cell expressed ELISA standards?
They more accurately correlate with cytokine levels in human serum or cell culture supernatants derived from human cells.

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Do I have to run all of my standards and samples in duplicate?
Yes, the duplicates are run in order to monitor assay precision and increase confidence in the assay results obtained. 

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Do I have to run all of my samples at one time?
No, each kit comes with single vials of each component, which can be aliquoted and stored. This allows the user to analyse different numbers of samples at different times, by coating only the necessary number of microtiter plate wells with the capture antibody.

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What type of reproducible results are obtained with the assays?
Each kit comes with a Package Insert containing a graph of typical data obtained. This data will vary according to the time and temperature of incubations. However the error bars on the graph indicate typical errors obtained in duplicate data points.

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What storage conditions are needed for a kit or product?
Each kit has a storage temperature stated on the label. Once kit components are reconstituted with water or the buffer indicated in the Package Insert, they should be stored frozen, between -20oC and -80oC, in small aliquots to eliminate the need for multiple freeze/thaw cycles.

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Is it possible to store the reagents other than indicated?
Storage of the kit components under conditions other than indicated is not recommended in order to assure proper performance of the test. 

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How should I store my samples?
Samples should be stored at -20oC or lower temperature. For long-term storage, it is recommended to freeze them at -70oC -80oC.

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How many samples will each kit run?
Each kit provides sufficient reagents to run at least fifteen 96 well microtiter plates, with 36 samples in duplicate, depending on the number of standards used for the standard curve.

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Can I modify the protocol?
Symansis' kits have been optimised to provide the best possible results. Modifying the format or protocol may give different results from those shown on the Package Insert.

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Do I have to run a Blank, or Zero Standard every time?
Yes, these are required for the calculations, and reflect any subtle but significant performance changes from day to day and assay to assay. They are also extremely helpful when troubleshooting the source of a particular assay problem.

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Can I alter the volume of sample I use in the assay?
It is not recommended that you alter the volumes since all Symansis' kits are designed for optimal performance at the given volumes

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What should I do if the reagent volume is not enough?
In cases of small reagent volumes, such as aliquoted reagents, centrifuge vials before usage to collect all the contents in the bottom of the vial.

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Can components from different kits be used?
Each kit contains components which have specific lot numbers to ensure that all of the components are performing optimally alone, as well as with all of the other components in the kit. QC testing is performed on these specific lots. It is never recommended to use your own components or components from other kits or vendors.

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Can I store the diluted standards and use them again?
No, we recommend that you use the diluted standards within the time required to perform the assay (usually one day).

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Do I make up my own standard curve?
Yes, you create your own standard curve, by diluting the provided standard, according to the kit's recommended range, in the Package Insert.

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How do you recommend I wash my plate?
If you are using an automated plate washer we recommend that the calibration be checked on a regular basis, and that the system is flushed with the Plate Washing Buffer prior to washing. The same is true for a manual washer. A repeater or a wash bottle can also be used. The user should be careful to ensure that all of the contents are aspirated and the plate tapped dry on lint-free paper.

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Do I need to use a plate shaker?
Reliable results can be obtained without a plate shaker, but the O.D.'s will generally be lower than those obtained using a plate shaker.

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Can I use an orbital flask shaker instead of a plate shaker?
An orbital flask shaker can be used if the microtiter plate is securely fastened and the shaker is set at low enough speed to ensure that the contents of the wells are not ejected.

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Why do I have to use wavelength correction between 450-570nm?
For the ELISA assay, reading at dual wavelengths is done to correct for the optical density contributed by the plastic well, the lamp and optical fluctuations.

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If I extract my sample, do I still need to follow the recommended dilutions given in the kit insert?
The amount of sample dilution needed after an extraction procedure will be affected by the effects of purification and concentration in the protocol used. The amount of dilution or concentration will have to be determined by the end-user.

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What is the expected concentration of analyte that I should expect to find?
The amount of a given analyte may vary not only from species-to-species, but also between tissue and cellular sources. The best source of this information is the current literature that is easily accessed through the Internet at multiple scientific databases.

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My optical densities were a little higher (or lower) than those in the Package Insert that came with my kit. Why?
The optical density is affected by a number of physical conditions such as time and temperature. We suggest that you shorten or lengthen the final incubation with substrate solution to compensate.

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Can Bovine Serum Albumin (BSA) be replaced by other things such as Fetal Bovine Serum (FBS), in the ELISA buffers?
Yes, the 1% BSA may be substituted with 10% FBS for plate blocking. Dilution of the standard, serum samples and detection antibody, in buffers containing 10% FBS may be preferable for testing of serum samples or tissue culture fluids.

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My samples are in tissue culture medium; do I need to dilute my standards in the same medium?
Yes, if you do not want to dilute the tissue culture medium with Assay Buffer. Your standards must be diluted into approximately the same medium as your samples. However, the use of media as diluents may result in a depression of your standard curve relative to the graph depicted in the Package Insert that you received in your kit.

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For how long can I store my capture antibody-coated plates?
ELISA plates that have already been coated with capture antibody can be stored at 4°C for a maximum of 3 days. During storage, the plates should be sealed and contain Blocking Buffer or Assay Buffer (e.g. 1% BSA in PBS or 10% FBS in PBS).

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What are the reasons for High Background?
Improper Washing: Insufficient washing, or the omission of one of the wash steps can lead to high background. Check volume of washing buffer reservoir and make sure all recommended washing steps are performed. Contaminated Substrate: Make sure there is no contamination of the substrate with metal ions or oxidising reagents, before use. Keep the extra substrate solution separately during the ELISA substrate development time. The substrate solution should not develop color by itself during this time. Contaminated Diluent: Most assay diluents are based on a protein matrix which can lead to the growth of microbes. Use only sterile pipettes to remove diluent needed from the storage container. Substrate exposed to light: Exposure to light may result in a blue color of the substrate. Keep solutions in the dark (vial) until ready to dispense into the plate. Wrong Conjugate dilution: Make sure the conjugate is diluted according to the guidelines in the Package Insert. Too high concentration of the conjugate might result in elevated background. Wrong Incubation Times/Temperatures: Generally follow the test protocol regarding incubation times and temperatures. However, if all wells are intensely and equally coloured with no intensity gradient observed in the standard dilution series, then it may be necessary to observe the substrate reaction as the colour is developing, in order to stop the reaction sooner. 

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What are the reasons for Incorrect Standard Curves?
Wrong reconstitution volume of lyophilised standard: Make sure the lyophilised powder is reconstituted in the correct volume and the correct buffer (deionised or distilled water with 0.05% NaN3). The reconstitution buffer and volume are given in the Package Insert. Reconstitution time too short: Make sure the reconstitution process is given enough time to allow complete solubilisation of the lyophilised powder. Wrong dilution of concentrated stock standard: Make sure the concentrated stock standard provided has been diluted according to the range indicated in the Package Insert. Wrong storage of stock material: Storage conditions of the stock standard solution is given in the Package Insert. Follow these guidelines exactly. Contamination of standard: Contamination of the standard should be avoided by usage of sterile pipettes and proper storage. Improper mixing during standard dilution steps: When preparing serial dilutions of the concentrated standard make sure there is proper mixing at each step of each dilution. Wrong measurement wave length: Make sure the plate reader used is adjusted to the correct wave length. Please check for the reference wave length. Standard from a different lot used: Use only the standard provided in the same kit to perform the assay. Do not substitute the standard from another kit. 

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What are the reasons for No Color Development?
Reagents not at RT at start of testing: Bring all reagents to room temperature before performing the test. Incorrect storage of individual reagents: Storage conditions of all kit components are given in the Package Insert. Follow these guidelines. Contamination of HRP-Conjugate: Make sure there is no contamination of the substrate before usage. Wrong dilution of HRP-Conjugate: If the concentrated conjugate is diluted to too low a concentration, color development will be weak. Follow the guide lines in the test Package Insert. 2 component TMB not mixed properly: If using a 2 component TMB substrate mixture, follow the ratio of mixture for the TMB substrate, as given by the supplier. Make sure the components are mixed properly before use. Omission of any incubation step: Make sure every step of the test protocol is performed. Microwells dried out after washing or during storage: After washing, tap microtiter plates on absorbent pad or paper towel to remove excess Wash Buffer. Use the microtiter plates immediately after washing or place upside down on a wet absorbent paper for no longer than 15 minutes. Do not allow wells to dry. Coating removed by scratching during washing/pipetting: Take care not to scratch the inner surface of the microwells when washing the plate or pipetting standards and samples. Samples not suitable or too dilute for the test: Check that the samples are suitable (e.g. compatable with test buffers) for the test or assay conditions and that they have not been over diluted. 

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What are the reasons for High Intra Assay Coefficient of Variation (CV) (data variation)?
Coating removed by scratching during washing/pipetting: Take care not to scratch the inner surface of the microwells when washing the plate or pipetting standards and samples. Cross contamination from well to well by peeling off plate cover: Carefully peel off the plate cover after incubation to avoid cross contamination from one well to another. Non-homogeneous samples: Make sure the samples you use for the test are homogeneous. Mix them before addition to the plate to avoid any gradients caused by freeze/thawing. Edge effect: Seal the plate during incubation and incubate on a shaker if possible. Pipettes used are not properly calibrated: Make sure the pipettes you use are properly calibrated. 

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When is it more appropriate to use a Chemiluminescent Immunoassay instead of an ELISA?
A Chemiluminescent Immunoassay is a more sensitive assay that is usually used only when samples contain very low concentrations of an analyte that are below ELISA detection levels. 

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What if I am not obtaining good results or am having difficulties?
Contact Us (or email tech@Symansis.com) to receive technical support.

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Why are Symansis products more sensitive standards in quantitative immunoassays involving native human proteins?
Symansis cytokines are expressed from human cells and have post-translational modifications that mimic naturally occurring human cytokines. Quantitative immunoassays such as ELISAs use antibodies directed towards a cytokine of interest to quantitate the sample. If the antibody was raised against a cytokine expressed from E. coli or CHO cells, the antibody may recognise a region that is normally glycosylated that is normally internalised when the protein is correctly folded. As a result inaccuracies may arise when using non-human cell expressed standards (such as those expressed from E. coli) to quantitate a natural human protein. Non-human cell expressed standards may over-estimate the naturally occurring cytokine if the antigen epitopes are absent on the standard protein due to a lack of the human specific post-translational modifications. Conversely, non-human cell expressed standards may underestimate the naturally occurring cytokine if the antibody is directed towards a region that is not normally exposed on the natural human protein. Symansis supplies ELISA kits with cytokines expressed from human cells to facilitate the generation of standard curves that more accurately quantitate the naturally occurring cytokine.



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