Peptide design and labeling
Pre-made antibodies
Secondary antibodies
Recombinant proteins
Custom production
Antibody applications
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Peptide Design & Labeling
1. What are the criteria for peptide antigen design?
Our criteria for peptide design are:
a) Hydrophilicity; to ensure the antigen is exposed on the surface of the protein.
b) Antigenicity; the ability of the peptide to be recognized by and interact with an antibody.
c) N- or C-terminal peptide; the termini are exposed in most proteins.
d) Contains MHC Class II preferred residues; such as D, Y, P, and F.
e) No homology with other proteins when searched for using BLAST.
f) Cytoplasmic domains are preferable for transmembrane proteins as they are more immunogenic.
g) Glycosylation and phosphorylation sites are avoided if possible.
h) No cysteine residue in the middle of the peptide; Cys is used to conjugate keyhole limpet hemocyanin (KLH).
2. Does Proteintech offer peptide labeling (e.g. fluorescein)?
Yes, we can label the peptide during peptide synthesis but we do not offer a standalone peptide labeling service.
We offer two options for peptide labeling:
a) Lysine-fluorescein labeling: The most efficient labeling is achieved by adding a pre-labeled lysine residue at one terminus of the peptide, either N-terminus or C-terminus. Alternatively, a pre-existing terminus lysine residue may be labeled with fluorescein. The end product is about 90% pure before HPLC purification, so HPLC purification becomes optional.
b) N-terminal-fluorescein labeling: The second method occurs at the last step of peptide synthesis. A fluorescein label is added to the free NH2 group in the N-terminus. The efficiency of this reaction is much lower than with lysine-fluorescein labeling so please keep in mind that the end yield may be low.
3. Can I include special amino acids like hydroxyproline in my peptide?
Yes, but there will be a surcharge of $200 for that particular amino acid. In addition, please let us know whether you need an L or D configuration on the residue.
4. What carrier proteins do you use and does the choice affect the price?
In most cases we use KLH as a carrier protein but you can also specify the use of BSA, ovalbumin, GST or MAP if necessary. The price will be the same in each case.
5. How is the carrier linked to the peptide?
The peptide is linked via a terminal cysteine (added by us) on the carrier protein.
6. How long does it take from peptide design to the purification of the antibody?
The toal length of time is approximately 4 months.
7. Is there a minimum length of peptide required for immunization?
The minimum peptide length required for a good immune response is 13 amino acids.
8. What is the maximum length of peptide you have synthesized?
We synthesize 40-60 amino acid long pepides regularly. Our record length is 83 amino acids long. It all depends on the particular sequence required and a judgment can be made once we have seen the sequence.
9. How many rabbits are immunized for each peptide service?
Each antigen will be injected into two rabbits.
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Pre-made Antibodies
1. How long will my antibody last if it is properly stored?
Our antibodies will remain functional for many years if properly stored.
2. How should I store my antibodies? Do I need to store it in aliquots?
Please store the antibody in the original tube at -20°C and do not aliquot. Our antibodies are stored in 50% glycerol which prevents them from freezing.
3. Could you send me detailed information for this antibody?
You can find the detailed information on our website. Please search and click the antibody of your interest; you will be directed to the datasheet for your antibody which includes validated applications, images, etc. If you would like to print the datasheet, simply click the PDF or print icon beside the Catalog No.
4. What is the concentration of my antibody?
The concentration of a given antibody can be found on the datasheet.
5. Can you provide me with the immunogen sequence of this antibody?
On the datasheet webpage of your antibody of interest, please click the link beside ‘Immunogen’ to view a description of the immunogen.
6. Is there a blocking peptide available for my antibody?
We use full-length recombinant proteins as immunogens to generate our antibodies. However, we do not test the neutralizing effect of the antigen (the recombinant protein) and do not have any other blocking peptides against our antibodies.
7. Has my antibody been tested on any species other than human?
We usually test antibodies on human and mouse cells or tissues. If you cannot find information associated with mouse cells or tissues or to find information on other species, please contact us.
8. Does my antibody cross-react with other species?
This is hard to predict with accuracy but will depend upon the extent of protein sequence similarity between the immunogen and the potential cross-reactive protein sequence. A pair-wise sequence alignment can be performed online through NCBI-BLAST website (Link to http://blast.ncbi.nlm.nih.gov/Blast.cgi).
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Secondary antibodies
1. How do I choose a secondary antibody?
When choosing a secondary antibody product, the following points should be considered:
a. Which animal was used to generate your primary antibody?
For instance, if your primary antibody is raised in a rabbit, you need to choose an anti-rabbit secondary antibody; if it is raised in a mouse, you need to choose an anti-mouse secondary antibody.
b. What class (or subclass) is your primary antibody?
The secondary antibody should match the class/subclass of the primary antibody. This is most important for monoclonal antibodies because different classes (or subclasses) can be found. Since polyclonal antibodies, such as those raised in rabbit, are usually of the IgG class, anti-IgG secondary antibodies are commonly used.
c. What application do you want to perform?
Enzyme-linked secondary antibodies such as those linked to peroxidase or alkaline phosphatase, are commonly used for ELISAs, immunoblotting and immunohistochemistry applications. For immunofluorescence and flow cytometry, which rely on fluorescence, a fluorochrome-conjugated secondary antibody is needed.
d. Is an adsorbed secondary antibody recommended?
In order to reduce non-specific background, secondary antibodies which have been adsorbed to serum of the host species are the first choice. For instance, if working with human tissues, cells or proteins, choose a secondary antibody that has been adsorbed with human serum or human IgG.
e. Is a F(ab’)2 fragment product necessary?
When working with some immune cells or tissues that have Fc receptors (B cells, leukocytes, thymus, spleen, blood, etc.), it is necessary to choose a F(ab’)2 fragment to eliminate non-specific binding through Fc receptors. Alternatively, you may perform an absorption step to block Fc receptors, using purified IgG from the host species of your secondary antibody.
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Recombinant proteins
1. What method does Proteintech use for recombinant protein purification?
We usually add 6x His tags or GST tags to the N-terminus of proteins and these proteins are then purified by Ni2+ and glutathione affinity chromatography respectively.
2. What storage buffers are used for your recombinant protein?
The His-tagged recombinant protein is usually stored in 1x PBS buffer (58mM Na2HPO4, 17mM NaH2PO4, 68mM NaCl, pH 7.4) containing 300mM imidazole and 10% glycerol.
The GST-tagged recombinant protein is usually stored in 1x PBST buffer (58mM Na2HPO4, 17mM NaH2PO4, 68mM NaCl, 1% Triton-X100, pH 7.4) containing 100mM glutathione (GSH) and 10% glycerol.
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Custom production
1. How should I choose between monoclonal and polyclonal antibodies?
a) Polyclonal antibodies are cheaper than monoclonal antibodies because they are much simpler to produce.
b) Polyclonal antibodies, which comprise a mixture of antibodies recognizing multiple epitopes, are more tolerant of minor changes in the antigen; but there is a higher chance of cross-reacting with other substances with a similar structure. In contrast, monoclonal antibodies are highly specific for one particular epitope on the substance of interest. Since monoclonal antibodies are so specific, they might not recognize the substance if it undergoes chemical changes or degradation.
2. Should I choose the long or short method for custom polyclonal antibody production?
The standard long protocol is our preferred method of production as it provides a better success rate than the short protocol. The short protocol is designed for customers who need the antibody quickly and are willing to trade success rate for delivery time.
3. I do not have any antigen; can Proteintech synthesize a peptide antigen or clone and express a full-length protein antigen?
Yes, we are happy to provide a comprehensive antigen production service.
4. Should I choose a full-length fusion protein or peptide antigen?
In our experience, full-length fusion protein antigens usually generate antibodies with both higher titer and higher specificity. We recommend the full-length fusion protein procedure if your protein of interest has low sequence similarity with other proteins. However, if you need an antibody that is specific to a certain protein whose sequence is highly similar to other proteins we strongly recommend using a peptide antigen. Other reasons you might choose a peptide antigen include: 1) Not having the cDNA for the protein of interest; 2) Expression of the full-length fusion protein has not been successful for some reason; 3) It has a comparatively low cost.
5. Is your custom antibody production service limited to human genes?
No, we do custom production of antibodies for proteins from any species.
6. How much antigen does Proteintech need for immunization?
For full-length protein antigen, the optimum amount needed is 1mg protein for each rabbit (2mg for two rabbits for each order). The minimum amount required is 300 μg/per rabbit. For peptide antigen, a minimum of 5mg of peptide conjugated to 2.5mg of KLH/per rabbit will be needed for immunization (In Proteintech's peptide antibody protocol, we use 10mg of peptide conjugated to 5mg of KLH).
7. What buffer should I use when sending the antigen to Proteintech?
The antigen can be stored in any biological buffer without detergent or urea. It can be sent frozen or at 4 °C at your discretion.
8. Can I send you antigen in gel slices cut from a SDS-PAGE gel?
Yes, protein in gel slices can also be used as antigen, although soluble or precipitated antigen is preferable.
9. My antigen aggregates in inclusion bodies or has to be denatured to prevent autocatalysis. Is it possible to perform immunization with antigen denatured in 8M urea?
Yes, it is possible. Proteintech have lots of experience of using antigens denatured in 8M urea.
10. How much plasmid is required for recombinant protein expression and purification?
10-20μg of plasmid is sufficient.
11. What kind of vectors should I provide for protein production or other related services?
We accept vectors that express GST or His tags. Please tell us the tag type, resistance of the vector, predicted molecular weight of the fusion protein and other related information when placing your order.
12. Can precipitated protein be used as antigen?
Yes. In many cases we have observed that precipitated protein yields more effective antibody than soluble antigen. It is thought that precipitated antigen provides a long- term antigen depot for continuing stimulation of the immune system and it may also be easier for antigen-presenting cells to engulf.
13. How much antiserum will I receive?
Each rabbit will yield approximately 12ml of serum in each production bleed and 45-50ml of serum from 90-100ml blood in the final bleed, totaling about 150ml of antiserum from two rabbits.
14. How does Proteintech determine which rabbit to purify serum from?
We decide that based on the ELISA results with the test bleeds. If both rabbits give good results, we will purify 15ml of serum from each rabbit. If one rabbit gives better results than the other, we will purify 30ml of serum from this rabbit.
15. How much purified antibody can I get from antiserum?
That depends on the concentration of the specific antibody in the serum provided. Usually we can get 2-10ml of purified antibody from 15ml of antiserum.
16. Do you offer multiple peptide immunization (immunize the rabbits with two peptides from the same protein)?
We can perform multiple peptide immunization. Please keep in mind that in some cases one peptide will be favored over the other for antibody generation.
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Antibody applications
1. I need an antibody suitable for matched-pair ELISA rather than indirect ELISA; can you test a specific antibody for this?
We would be pleased to investigate this for you. Please contact us for more detailed information.
2. I have purchased an antibody that will be used for IF and/or IHC. Do you offer a negative tissue control for this antibody?
Yes, we can offer you a negative tissue control. Please contact us for price information.
3. Are your antibodies suitable for immunohistochemistry on frozen sections?
Our antibodies have only been tested on paraffinized tissue sections; we do not test their performance on frozen sections.
4. I find that the observed molecular weight of my protein in a western blot is different from the predicted molecular weight. Why might this be the case?
Please keep in mind that the predicted molecular weight is calculated from the protein sequence. There are several possibilities which may explain this observation:
a) Degradation of the lysate: The sample may have been degraded by proteolysis; explaining why it will show a band with a different molecular weight than the one predicted from the sequence.
b) Protein modification: Some proteins, such as membrane proteins, are commonly modified (e.g. glycosylation) after translation. This may result in the protein having a different molecular weight to that predicted from the primary protein sequence.
5. I find that there are additional bands in the western blot result other than the major band. What might these bands be?
There are many factors that are responsible for additional bands appearing in western blot results:
a) Mechanical degradation of the lysate: If samples are stored for a prolonged period or if proteins or membranes are fractionated after homogenization of the starting tissue, additional bands which are faster migrating than the targeted one may appear. To get around this use fresh lysate.
b) Proteolytic degradation of the lysate: Proteolysis is a common cause of the appearance of background bands. Make sure there are sufficient protease inhibitors in the sample.
c) Too much protein sample was loaded
d) The detection system used was too sensitive
e) The western blot membrane was not blocked correctly.
f) Homologous proteins or other isoforms of the target protein: Around one third of proteins belong to protein families, containing homologous proteins which may cross-react with antibodies. Furthermore, one single gene can be translated into isoforms with different molecular weights. Antibodies usually can detect more than one of them.
g) Unspecificity of the antibody: Although Proteintech applies the most stringent quality assurance standards to ensure that our antibodies do not bind multiple targets; it is possible that the antibody binds some unrelated proteins, especially on those lysates which we did not test.
6. I cannot detect my antigen after western blotting?
The concentration of antigen may be too low. If the relative concentration of the antigen of interest is too low (less than 0.2 % of total protein), it may be difficult to detect. If this happens please load more sample, or enrich the antigen by fractionation or by immunoprecipitation.
7. What type of membrane do you suggest for Western blotting, i.e. polyvinylidene fluoride (PVDF) or nitrocellulose?
We usually suggest customers using PVDF membranes rather than nitrocellulose membranes, because PVDF membranes offer better protein retention, physical strength and broad chemical compatibility. The typical binding capacity of commercially available PVDF membranes is 100-200 μg/cm2, compared to that of nitrocelllulose membranes having a binding capacity of 80-100 μg/cm2. However, if the detection requires that the transferred protein retains its native conformation after transfer, for instance, when the antibody recognizes 3-D structure (e.g. an antigenic epitope consisting of non-contiguous residues), nitrocellulose membranes might be preferred.
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