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Technical Resources |
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1: What is the difference between monoclonal and polyclonal antibodies? 3: What host animals are available? 4: What type of antigens can be used? 5: How many animals are immunized? 6: How many clones are included? 7: What yield of product is delivered? 1: What is the difference between monoclonal and polyclonal antibodies? 2: What host animals are available? 3: What type of antigens can be used? 4: How many animals are immunized? 5: Are there any options if the standard protocol produces low titres? 6: What are the yields of antisera for type of bleed in the different host animals? 7: How do I select an appropriate peptide sequence? 8: What are the advantages to using synthetic peptides as antigens? 9: What peptide purity do I require? 10: Will my peptide be difficult to synthesize? 11: How long will it take to make an anti-peptide antibody? 12: Which carrier molecules are available, and which should I use? 13: Should I use a MAP (multiple antigenic peptide)? 1: What applications require purified antibodies? 2: Which purification procedure should I use for my antibody? 1: Should my antigen be cross-linked to a carrier protein? 2: How do I select a carrier molecule for my peptide? 3: Why is my KLH-conjugated peptide solution turbid in appearance? 4: What technique should I use to conjugate my antigen to a carrier molecule? 5: Should I label my primary antibody for direct detection? 6: What should I use to label my antibodies? Answers - monoclonal antibodies - back to FAQs 1: What is the difference between monoclonal and polyclonal antibodies? Monoclonal antibodies are produced from clones of a single hybridoma cell and as a result, recognize only a single epitope on the antigen and have a defined specificity. Polyclonal antibodies recognize independent epitopes.
Hybridomas are cells generated by fusing splenocytes and myeloma cells, which are screened to produce antibodies against the antigen used for immunization.
3: What host animals are available? SAS currently only uses mice as hosts for monoclonal antibody production
4: What type of antigens can be used? Clients can provide proteins and haptens (either conjugated or unconjugated peptides). The antigen can be provided lyophilized, in solution, or in an SDS/PAGE gel.
5: How many animals are immunized? Our standard hybridoma development protocol utilizes 4 female Balb/c mice.
6: How many clones are included? In a typical hybridoma project, X clones are delivered to the client.
7: What yield of product is delivered? Although yields can never be guaranteed, on average we recover X ml of Y mg/ml for every mouse used. Answers - polyclonal antibodies - back to FAQs 1: What is the difference between monoclonal and polyclonal antibodies? Monoclonal antibodies are produced from clones of a single hybridoma cell and as a result, recognize only a single epitope on the antigen and have a defined specificity. Polyclonal antibodies recognize independent epitopes.
2: What host animals are available? SAS currently uses mice and rabbits [female New Zealand Whites (NZW)] as host animals for polyclonal antibody production, and will include rats, guinea pigs, chickens, and goats by December 2005.
3: What type of antigens can be used? Clients can provide proteins and haptens (either conjugated or unconjugated peptides). The antigen can be provided lyophilized, in solution, or in an SDS/PAGE gel.
4: How many animals are immunized? The client decides the number of host animals required depending on the volume of antigenic serum require. Typically, when mice are used as a host X-Y animals are used, whereas 1-3 rabbits is often sufficient.
5: Are there any options if the standard protocol produces low titres? If titres are insufficiently low, the standard protocol can be extended with additional antigen injections. Contact us for specifics about our extra antigen boosting schedule.
6: What are the yields of antisera for type of bleed in the different host animals?
7: How do I select an appropriate peptide sequence? Although the design of peptides for use as antigens is not exact, several hints can help maximize the likelihood of success in producing antibodies. It is important to select sequences which are found on the surface of the native protein, and contain residues that are hydrophilic and have flexible side-chains. A BLASTp search should be performed and a sequence with minimal homology selected to reduce the chance of non-specific antibody binding. Also, peptide sequence length, hydrophobicity of residues, and specific sequence issues are critical. See our peptide design page for more detailed suggestions.
8: What are the advantages to using synthetic peptides as antigens? One of the main advantages to using synthetic peptides for antibody production is that specific epitopes can be targeted. Peptides can be designed and modified to contain precisely what is required for your experiment. Synthetic peptides are also relatively inexpensive to produce and can be obtained in a highly purified form.
9: What peptide purity do I require? For use as an immunogen, peptide purity (expressed as the % of correct amino acids in the peptide) should be at least 70%.
10: Will my peptide be difficult to synthesize? Several factors will influence the production and purification of a peptide including its length and amino acid composition, hydrophobicity and stretches of the same residue. Our experienced protein chemists will help review your sequence to determine whether the peptide can be synthesized at sufficient purity for use as an immunogen.
11: How long will it take to make an anti-peptide antibody? We provide an extremely fast turn around time from the receipt of a clients' order to delivery of the antibody. Peptide design and synthesis is generally complete after 10 days, and our standard polyclonal antibody protocol requires 70 days, so our clients should receive their custom antibody after 2.5 months.
12: Which carrier molecules are available, and which should I use? Conjugation of a peptide to a carrier molecule is critical since peptide molecules alone often fail to initiate an immune response. Carrier molecules contain multiple epitopes that can stimulate T-helper cells, which lead to a B-cell response. Keyhole-limpet hemocyanin (KLH) and bovine serum albumin (BSA) are the most common protein carriers, and KLH is preferred since it is more antigenic in the majority of animals. Also, BSA is often used as a blocking reagent in assays, thus an anti-peptide antibody raised against a BSA conjugated peptide will show some specificity towards the buffer reagent. This can result in a false positive signal.
13: Should I use a MAP (multiple antigenic peptide)? MAP is simply an alternative to KLH conjugation. It is recommended only for short peptides located either internally within a protein or at the N-terminus. KLH conjugation is recommended for a peptide at any location in a protein and when there is a cysteine residue within the peptide sequence. Answers - antibody purification - back to FAQs 1: What applications require purified antibodies? Several advanced antibody applications require highly purified antibodies including immunohistochemistry, immunocytochemistry, immunoassays, immunoblots, and immunoaffinity purification.
2: Which purification procedure should I use for my antibody? The purification protocol and reagents used will depend upon the intended use of the antibody, the subclass of immunoglobulin, and the species in which it was raised. SAS offers a custom consultation to determine the correct choice of purification methodology for our clients. Answers - peptide conjugation - back to FAQs 1: Should my antigen be cross-linked to a carrier protein? Certain types of antigens (peptides and haptens) are insufficient immunogens when injected alone. These types of molecules should always be conjugated to a carrier protein when used for antibody production.
2: How do I select a carrier molecule for my peptide? The carrier molecule you select will depend on the species you are studying. Keyhole limpet hemocyanin (sea snail hemoprotein) is most often used for vertebrate research because there are no homologous vertebrate proteins resulting in little if any non-specific antibody activity. For invertebrate studies, it is best to use a carrier such as bovine serum albumin or ovalbumin.
3: Why is my KLH-conjugated peptide solution turbid in appearance? Keyhole limpet hemocyanin (KLH) conjugated peptides often have limited solubility in water due to its size and structure. KLH is a large (MW = 1x105 to 1x107) protein, however, this does not affect its immunogenicity and cloudy solutions can be used for immunizations.
4: What technique should I use to conjugate my antigen to a carrier molecule? Many different protein cross-linking techniques are available, and selecting one will depend on many factors. The coupling reaction should produce a conjugate that resembles the native protein, thus factors such as the location of a peptide sequence within a protein will determine where the carrier should be linked. Also, the chemistry of the immunogen must be considered since different techniques require specific chemistries to function. Common coupling methods will link carriers to antigens to free amino, carboxylic acid, or sulfhydryl groups.
5: Should I label my primary antibody for direct detection? Labelling you primary antibody for use in direct detection can reduce the number of steps and washes in your procedure and is less prone to background that indirect methods. However, direct detection can be less sensitive than indirect methods since chemistries used to label your primary antibody can reduce its activity. The choice of direct vs. indirect detection methods depends on your specific experimental requirements.
6: What should I use to label my antibodies? There are numerous labels for your antibodies and selecting one will depend on your requirements since each has their own advantages and disadvantages. Biotinylated or enzyme-linked antibodies are often useful for immunohistochemistry, immunoassays, and immunoblots. Fluorophore labelled antibodies are useful in immunohistochemistry if a confocal microscope is available, and extremely useful for immunocytochemistry and flow cytommetry. | |||||||||||||||||||||||||||||||||||||||||||||||||||||
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