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技術問答

  • Is there any species preference for primary antibodies for western blot?
    It does not matter from which species the primary antibody comes from for a western blot application. There are many good secondary antibodies available for most species. What is important is which part of a protein an antibody is made to. Is it going to recognize epitopes (stretches of 5-6 amino acids), which are exposed on a given protein after transfer to a membrane? Depending on the applied condition, some proteins can refold following the transfer, which can make a given epitope inaccessible. It also depends on if the western blot is performed in native or denatured conditions. The addition of 6-8 M urea to your loading buffer might help to keep your protein unfolded and accessible for an antibody to bind. Before you use an antibody in western blot, please check to which part of the protein it was made. Are there any references confirming the use of this antibody for this specific technique?
  • If an antibody works in IL, will it also work in WB and vice versa?
    It depends on the antibody binding site on the protein. Is this part exposed after fixation or following the western blot? If a polyclonal antibody is made to a synthetic peptide, it recognizes a pool of epitopes (linear epitopes are streches of 5-6 amino acids). If such epitopes are not exposed following fixation, the antibody is not going to work.
  • If an antibody detects a protein in one species, will it work for all other species?
    Not really, it all depends on the conservation level of the specific peptide used to elicit this antibody. It can be checked by comparing the peptide used to elicit the antibody in question, to the sequence of your protein. The conservation level between the peptide used to make an antibody, and the peptide found in your protein seqence, needs to be around 70% to allow an anti-peptide antibody to work, but may be lower for antibodies made to larger parts of a protein. In some cases, 6-8 M urea needs to be included to fully unfold the protein. The electrophoretic mobility of a protein can also vary between different species.
  • How do I know if an antibody will work on my species and in my application?
    Yes, to a certain extent it is possible to predict the outcome before making actual experiments. What is worth checking is the conservation level of the peptide or protein sequence used to elicit a given antibody, to the protein you are planning to detect. If a sequence is not provided on our website, please send us an inquiry about this, together with the sequence of the protein you are aiming to detect.
  • How can different results using the same antibody be explained?
    There can be different reasons behind it: You obtained another batch in the second purchase. Please, check it on the tube which contains a specific lot number. Each antibody batch is unique and can vary in its binding properties. Another secondary antibodies was used. Another reagent was used. Another set of samples were used.
  • Why do I get a lot of background bands in my western blots?
    Have you checked the recommended conditions to use for this specific antibody, included on the product info sheet? Some antibodies will work reasonably well and give good results regardless of the applied conditions, like membrane type, load per well and developing reagent. Some antibodies require a bit more consideration. Less background can be obtained for some antibodies when using a PVDF instead of nitrocellulose membrane for transfer, or by applying the recommended blocking. There are also variations between secondary antibodies. However, the most important factor to consider when evaluating the western blot results, is often the type of sample being analyzed. For more information, you can either contact us.
  • Can I reuse my primary antibody solution?
    When attempting to do quantification, it is generally not recommended to re-use an antibody solution, as this may give non-consistent results. Therefore, such information is not included in our product info sheets. Every antibody is different, and the re-use approach might work for one antibody, but give weaker and weaker results for another. This is due to a certain amount of antibody being depleted in every performed incubation. You need to try this approach in your particular set-up to know if it is viable. But please keep in mind that the results might not be consistent. If you would like to save your primary antibody, you could consider using a more sensisitive detection reagent instead.
  • How long do antibodies last, and how do I store them?
    Each antibody is different. Some might last for 20-40 years, while others may only last a year. Storage conditions are of importance to extend the antibody lifespan, but the stability of each specific antibody might be different. Therefore, please follow the information provided on the specific product information sheet for each antibody. What works for one antibody, might not necessarily be applicable for another. This is very important to keep in mind. If iyou have further questions, please contact us.
  • What's the difference between serum, total Ig fraction and purified antibodies?
    One big difference between antibodies offered in the formats serum, total immunoglobulin fraction and antigen-purified, is the amount of specific antibodies per µl or µg. When calculating how many experiments an antibody can be used for, one should always check the suggested antibody dilution in the context of amount of protein/well in Western blot, not how many µl or µg of antibody is offered in one tube. Below is a table showing the amount of specific antibodies in different formats.
  • What are the advantages of polyclonal antibodies?
    Polyclonal antibodies will recognize a mixture of different epitopes of an antigen and are more tolerant to small changes in the nature of it, like polymerization or slight denaturation. Polyclonal antibodies are the preferred choice for detection of denaturalized proteins.
  • What is the concentration of the specific, polyclonal antibodies in serum?
    Most commonly, the concentration of specific antibodies in serum varies between 0.05-0.2 mg/ml of serum. In special cases, strong polyclonal serum can contain up to 1 mg of specific antibodies per ml of serum.
  • What are the advantages of monoclonal antibodies?
    Monoclonal antibodies recognizes only one chosen epitope of the antigen, which can be a disadvantage in some assays, such as immunoprecipitation, and when making immunoaffinity columns. A monoclonal antibody is as good as a primary antibody in an assay for detection of antigen in a tissue. Theoretically, they should give much lower background than polyclonal antibodies, however that is not always the case. Monoclonal antibodies have high homogeneity and give highly reproducible results (if other experimental conditions can be kept constant).
  • What is important when choosing a peptide for immunization?
    Usually the C or N-terminal of the protein is used, as those parts of the protein are exposed. Also, to mimic protein behavior, the synthesized peptide should have similar structure and charge as the protein it has been "cut out off". Therefore: Peptides derived from the C terminal should have N terminal modified by acetylation Peptides derived from the N terminal should have C terminal modified by amidation Peptides derived from an internal sequence should have both ends modified The following points should also be considered: Are there any other proteins from the family of interest, where cross-reactivity should be avoided? Is the crystal structure of the protein (or homologous protein) known? This would be helpful for the peptide chemist in searching for the best peptide for antibody production. What is the final application of the produced antibodies? Native or denatured techniques?
  • What kind of antigen can be injected?
    Immunization can be done using native proteins, recombinant proteins, peptides, carbohydrates or other compounds of microbial, fungal or viral origin. Minimum molecular weight needed to induce sufficient immune response is 5-10 kDa. Biohazardous materials for immunizations are not accepted. Important notes: If the antibodies are going to be used on the denatured target protein (example: Western blot, immunohistochemistry on fixed tissues), denatured forms of antigens are preferred (protein in inclusion bodies). If the antibodies are going to be used on native target proteins (example: immunoprecipitation), non-denatured forms of antigen are preferred (protein in solution free of denaturing agents). Not all peptide antibodies will recognize native protein, therefore a careful choice of peptide sequence is of crucial importance. Antibodies made against recombinant proteins expressed in bacteria can in some cases fail to recognize native protein. The reason for this might be incorrect folding of the protein antigen when expressed in bacterial cells. No guarantees can be given in advance for a success of any immunization program. Recommended references on the subject: "Monoclonal antibodies: principles and practice" by James W. Goding, 1996, ISBN 0-12-287023-9; Publisher: Academic Press. Using Antibodies: A Laboratory Manual, E. Harlow and D. Lane, 1999, ISBN: 0879695447; Publisher: Cold Spring Harbor Laboratory Press. Hjelm et al. (2012). Parallel immunizations of rabbits using the same antigen yield antibodies with similar, but not identical, epitopes. PLOS ONE.
  • How much antigen is required for immunization?
    It depends on the immunogenicity of the antigen. In a standard protocol (for rabbit, goat or hen) you may use around 500 µg of peptide/animal/15 week program, or around 400 µg of protein/animal/15 weeks program. Lower amounts of antigen (less than 10 µg) are acceptable in cases of low relatedness between the antigen and proteins of the animal chosen for immunization.
  • Why will some antigens only induce response resulting in IgM antibodies?
    Responses against highly conserved mammalian proteins are often weak and mainly resulting in IgM antibodies, owning to lack of stimulation of T cells (Goding 1993). However, there are also exceptions. In cases of conserved mammalian antigens, it is beneficial to use a species which is more evolutionarily distant e.g. hens.
  • How should the antigen be prepared for immunization?
    Antigens should be supplied in PBS or carbonate buffer, at physiological pH. If isoelectric point of a protein lays at physiological pH, it will become insoluble. For keeping protein soluble, pH of a buffer should be adjusted below or above. Addatives should be avoided. Insoluble antigens (e.g. inclusion bodies) can also be used for immunization. However, one must keep in mind that other proteins will often also be present in inclusion bodies, not only the target protein. The desired antigen concentration is 1 mg/ml, but lower concentrations are also acceptable.
  • Which species to choose for immunization?
    Advantages of using both IgG (rabbit, goat) and IgY (hen) antibodies developed against the same antigen: Independent confirmation that the expected target protein is detected. Antibody pools with distinct properties, complementing each other in different techniques (Western Blot, immunoprecipitation etc.), making double staining possible. A ground rule for immunization is to choose an animal that is genetically distant from the antigen source (e.g. hens are very suitable for production of antibodies against conserved mammalian proteins).
  • Are antibodies made in rabbit better than those made in chicken?
    Antibody production outcome is a combination of the an antigen, the specific animal immunological response, and testing. For certain proteins, like conserved mammalian targets, antibody production in chickens is recommended. Chickens are more evolutionary distant, and their immune system will respond better to conserved mammalian proteins, which might not be recognized by the immunological system of a goat or a rabbit.
  • If my antibody works in immunolocalization, will it also work in a western blot?
    It depends on which part of the protein the antibody is recognizing, and how this part is exposed after fixation or following the western blot. If a polyclonal antibody is made to a synthetic peptide, it is recognizing a pool of epitopes. If such epitopes are not exposed following fixation, the antibody will not work. In some cases, addition of 6-8 M urea to the loading buffer can help to unfold the protein.
  • How to store antibodies?
    Antibodies present in serum Serum is a very stable format for antibody storage. In -20°C or -70°C, serum can usually be stored for years. In some specific cases, the shelf-life can be shorter for anti-peptide antibodies. For very short periods of time, serum may be stored at + 4°C. In some cases, more careful freezing with a first step at -20°C, followed by -70°C is beneficial. Total IgG fraction (IgG antibodies purified on Protein G matrix) Generally, protein G purified antibodies are stable. They can be stored in -20°C or -70°C for years. For short-term storage, add some azide to a final concentration of 0.02 % (or another preservative). Total IgY fraction (IgY antibodies purified by precipitation from egg yolk) Purified IgY fractions are very stable, even at room temperature (although we do not recommend it as storage conditions). IgY can be stored at + 4°C with 0.02 % sodium azide (note: azide inhibits activity HRP enzyme) or gentamicin sulfate (50 µg/ml). Avoid freezing and thawing of IgY, and storing it on dry ice. IgY antibodies can be stored at -20°C. "The IgY preparations were stable over time. No loss of antigen recognition was observed after storage for 3 years at + 4°C". F. De Ceunick et al. Journal of Immunological Methods 252 (2001) 153-161. Egg yolk Antibodies in egg yolk should be stored at 4°C with 0.02 % sodium azide (note: azide inhibits activity HRP enzyme) or gentamicin sulfate (50 µg/ml). Egg yolk should NEVER be frozen as this will complicate purification of the antibodies. After 6 months of storage, purifying antibodies present in egg yolk might be somewhat difficult. Affinity purified antibodies Affinity purified antibodies are the most fragile. Caution should be taken when considering storing conditions, which should be checked experimentally for every single antibody. Affinity purified antibodies against different epitopes can vary in stability. Some will precipitate directly after the purification, while the activity may still remain. It is difficult to predict storage conditions for a given antibody in advance - there are some alternatives to be tested: -20°C or -80°C +4°C with preservatives like azide (0.02%) or merthiolate -20°C with glycerol at a final concentration of 10 or 50% -20°C with BSA at final concentration of 0.05-0.5% IgG Mammalian, polyclonal antibodies can precipitate following affinity purification. This can occur directly after purification, or overnight during cold storage. Some antigens will stimulate the production of a class of IgG, called cryoglobulins, which precipitate at low temperatures. Heating the cryoglobulins up to room temperature can solve this problem. The antibody solutions can also be centrifuged to remove precipitates. IgY Chicken antibodies can also precipitate when stored in the cold, wither directly overnight, or after several weeks. Heating the IgY up to room temperature often helps to dissolve those precipitates. Otherwise, IgY solution can be centrifuged to remove precipitation prior to use. Antibody solutions stored without preservatives are at the risk of being contaminated by bacterial growth, which is one of the most common reasons for protein inactivation. General recommendations: For larger volumes of affinity purified antibodies, filter-sterilize the antibody sample and aliquot the solution to avoid multiple freezing and thawing cycles. Ideal storage occurs at protein concentrations around 0.5-1 mg/ml. In cases of IgM antibody production, check protein stability in different storage conditions. Important note: Sodium azide will inhibit horseradish peroxidase, as well as interfere with some coupling methods and biological assays. However, the amount present in IgY preparation (0.02 %) can be washed away in ELISA or Western Blot when IgY is used as primary antibody at dilutions of at least 1:2000. Alternative agents for preventing bacterial growth in antibody solution: Thimerosal at 0.01% Gentamicin sulfate at 50 µg/ml
  • How do I choose peptide endings appropriate to my studies?
    The choice of peptide endings often falls simply to the question: am I happy with the default of a free amine group at the N-terminus of the peptide, and a free acid group at the C-terminus of the peptide, or do I need something different? A good example of where something different is needed, is where the peptide is a natural hormone or an analog of a natural hormone with an amide group at its C-terminus, such as Substance P. In a case like that, the only reason to vary from having the peptide made with a "natural" amide C-terminus would be to examine the effect of placing an unnatural free acid group on the C-terminus. Another common situation is where the peptide represents an internal portion of a protein sequence, i.e. does not arise from the natural N-terminus or C-terminus of a protein. It may be best in such cases to "block" the ends of the peptide, such as by having the C-terminus made as an amide, and having the N-terminus blocked by acetylation. The peptide would then be indicated as having the structure: Acetyl-(Peptide Sequence)-amide. By using this design, the following is achieved: It avoids the unnatural introduction of a charged group (amine or acid) at a site in the peptide, where the same site in the parent protein has no such charged group. Blocking the ends like this can be beneficial because the peptide is then more likely to behave like, or be recognized, as if it were a part of the whole protein from which the sequence was chosen. The peptide will be more resistant to breakdown resulting from the actions of exopeptidases, which attack the peptide from the ends. It would then persist longer in the bioassay system, such as an assay involving living cells or biological extracts which contain peptidases. Not all effects of blocked ends are helpful, however. Blocking charged groups on the ends of a peptide decreases the solubility of the peptide, maybe to the extent where solubility becomes a limitation on the effective concentration of that peptide which can be obtained in solution. It may even make the peptide so hard to dissolve that its usefulness is compromised. Thus, a decision may need to be made about what is more important: solubility, or the closeness of a peptide's structure to resembling the protein from which its sequence was derived? Similar issues arise each time a new peptide study is devised. For example, in the study of cytotoxic T cell epitopes, it is appropriate to have short peptides made with free amine and free acid endings, because they are the natural endings of peptides which have been processed intracellularly from whole proteins. Natural helper T cell epitopes are longer than cytotoxic T cell epitopes, and even though in nature helper T cell epitopes have free ends, relatively short end-blocked synthetic peptides may function better in helper T cell assays than peptides with free ends. The reason may be related either to the synthetic peptide not being made with an ideal length, or it may be related to the amount of time the peptides persist in cultures before being broken down. The lengthened amount of time that end-blocked peptides have, to begin exerting a biological effect in a culture, may be more important than their ability to exactly mimic a natural epitope. Other endings which may be chosen for specific purposes include: Adding a biotin group at one end of a peptide (usually the N-terminus) as a convenient way to capture the peptide via a high affinity noncovalent interaction; Adding a Cysteine residue at one end of a peptide to allow covalent capture under mild conditions; or Adding a fluorescent group, to allow tracing of the peptide.
  • How long would it take to raise antipeptide antibodies?
    There are two immunisation schedules available, a 9 week immunisation protocol or a 12 week immunisation protocol. The peptide conjugates require 2-3 weeks for preparation so this amount of time should also be included into the target delivery time for your antibodies. Please contact us for more specific details and costs for our antibody projects.
  • What purity is offered for custom synthesized peptides and how is this purity characterised?
    Yao-Hong offers >70%, >80%, >90% and >95% purity for our customs synthesised peptides. The purity is determined by analytical HPLC. We also include MS analysis as standard. We can also offer LCMS if required.
  • What are MAPs and how can they be useful?
    MAP stands for Multiple Antigen Peptide and is a Lysine branched peptide (2, 4 or 8 branches) which can be used to raise antibodies . The advantage of these peptides is that they can be designed to incorporate both B Cell and T Cell epitopes and do not require conjugation to an immunogenic protein carrier suchas KLH.
  • Is there any way of determining which region of a protein will be favourable for raising antibodies?
    There are various programs, including both internet and in-house programs which can be used to identify regions within a protein that may be more favourable for the raising antipeptide antibodies. Our in-house program called Preditop is based on secondary structure determinations and have proved to be very helpful in identifying suitable regions in proteins which have resulted in successful polyclonal antibodies.
  • I require a fluorescent label peptide, which fluorescent labels do you offer?
    We offer many labels which can be covalently attached to peptide to ensure stability. Commonly requested “green” labels include 5,6-carboxyfluoresceine and 5,6-carboxytetramethylrhodamine as a “red” label.
  • What are pseudoproline amino acids and what advantage do they have in peptide synthesis?
    Pseudoproline dipeptide reagents are special secondary structure disrupting amino acid reagents based on serine and threonine. They stop the peptide forming secondary structure whilst the peptide is growing on the solid phase. This is thought to be one of the main issues with successful peptide synthesis. On cleavage the native sequence is re-established.
  • What are the typical contaminants associated with peptide synthesis?
    For purified peptides, the main contaminants are deletions and truncations of the parent sequence. This occurs when the reaction efficiency of the incoming amino acid is reduced due to factors such as steric hindrance and secondary structure of the growing peptide chain. A truncation is a C-terminal fragment of the peptide which is shorter than the parent. A deletion is the parent sequence which I missing one or more amino acids in any part of the peptide. Certain reagents which are used in the work up to cleave the peptide from the solid phase may also carry over, however, these are volatile and evaporated in the freeze-drying process and removed during the purification step.
  • What is the difference between peptide purity and peptide yield?
    Peptide purity is determined using analytical methods such as RP-HPLC and MS analysis. It provides a percentage of the sample which is detected using UV absorbance of certain chemical moieties (for HPLC) or charged species in the case of MS. It does not identify the contribution of water and salts in the sample, for example. Amino Acid Analysis is used to calculate the yield or actual amount of peptide only in the peptide sample. In this way, the moles of peptide can be calculated and the difference between the weighed mass (before AAA) and the measured result (after AAA) is normally attributable to water and salts.
  • What are the typical salts associated with my peptide?
    If no acetate exchange was performed then the dominant ion will be trifluoroacetate, one for each basic amino acid and the N-terminus. If you prefer an alternative salt, we can offer an acetate exchange using acetic acid, as an example.
  • If I have biotin in my peptide, do I need a spacer between the biotin group and the peptide sequence and if so which spacer should I use?
    Yes, we recommend a spacer between the Biotin and your sequence to ensure adequate spatial separation between the two moieties to ensure that the sequence is accessible to the binding partner. Different types of spacers can be used, please contact usfor more information.
  • What can I do to increase the cell permeability of a peptide?
    Sequences which exist in the public domain such as penetratin and the TAT sequence can be added to your sequence. These motifs are known to assist in the transportation of peptides across the membrane and into the cell. Please contact usand we can advise how to best incorporate these sequences into your peptide to improve synthesis outcome.
  • How do I characterize batches of my "drug candidate" peptide, which contains a D-amino acid?
    There are many analytical techniques available and we have listed below the most common ones, with some comments: Mass spectrometry (MS) is one of the most powerful techniques for checking that the "molecular formula" is correct, i.e. the peptide contains the stated amino acids. MS also can be used (on the right machine) for confirming the amino acid sequence of the peptide, by fragmenting the peptide within the instrument and analysing the fragments. This technique still does not readily distinguish stereoisomers of the "correct" sequence (in this case, enantiomeric peptides). Analytical reverse phase HPLC is a very sensitive technique for detecting differences in physicochemical properties between similar peptides, and has the potential to act as a quality check method for whether or not amino acids of the correct stereochemistry were used to make the peptide. It requires the use of well-characterized standards, or can be used to get a measure of "relative" properties (differences) between test samples. However, identical elution of two samples in analytical HPLC is, in itself, NOT proof that the samples are identical. High pressure liquid chromatography, combined with continuous mass spectrometry of the eluate from the chromatography column (i.e. LC/MS) combines the power of HPLC and MS to give a more detailed picture of the components of a mixture, but may not be useful if the material is homogeneous by HPLC. Polarimetry (optical rotation of polarized light) is a simple technique for verifying that the peptide has the expected optical properties (a function of its stereochemistry), but polarimetry may not have enough sensitivity to detect a change in a single amino acid. N- or C-terminal amino acid sequencing is a good way to ensure the amino acids were assembled in the correct order, but is rarely necessary because a similar assurance can be obtained from comparative analytical HPLC (by comparison with verified standards), fragmentation mass spectrometry, NMR etc. 6. Amino acid analysis is good for ensuring the correct amino acids are present, and in the expected ratios, and is also one of the best ways to measure the true "peptide content" (i.e. it does not measure counterions, solvent and nonpeptide impurities). It is not a good way to detect amino acids with incorrect stereochemistry (i.e. L- versus D- isomers). Depending on the size of the peptide, 2-D proton NMR may be suitable for setting an absolute characterization standard for your peptide, but it may require a lot of development work by a chemist to assign an identity to each signal, and therefore to confirm that the data shows the structure is exactly what you expected it to be. Chiral column HPLC is another way to separate peptides of identical amino acid sequence, differing in stereochemistry of one or more of the amino acids. A chiral column can also be used to check the stereochemistry of the individual amino acids after hydrolysis of the peptide. Capillary electrophoresis is a very sensitive technique for testing the homogeneity of a peptide, and could also pick up mistakes in the assembly/stereochemistry of the peptide, by comparison with verified standards. Other supplementary methods would include circular dichroism, bioactivity measurement, immunoreactivity (recognition by a specific antibody), elemental analysis, and spectrophotometry. Testing for impurities, in contrast to checking for identity, would make use of the techniques above in different ways. The presence of "unexpected" peaks or signals would be evidence for impurities; however, the absence of unexpected peaks or signals would not be proof of the absence of impurities. More content in preparation (check back soon), for example: Isomerization Bioactive impurities Batch variation Modified peptides and unnatural amino acids.
  • How do you calculate hydrophobicity?
    The hydrophobicity algorithm used is from Fauchere & Pliska (1983), Eur J Med Chem 10:369. The parameters for the amino acids are shown below. Peptide endings are also taken into account in calculating the hydrophobicity value. "Hydrophobicity" varies between approximately -1 (most hydrophilic) and +2 (most hydrophobic). A second hydrophobicity parameter, "Hydro-2", is calculated as the criterion "percentage of hydrophobic residues".
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