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Protein Conjugation |
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SACRI antibody services (SAS) have highly skilled scientists dedication to antibody and protein conjugation. SAS staff have over 20 years experience in protein chemistry and cross-linking techniques. Whether you wish to link a peptide antigen to a carrier molecule for immunization or wish to label antibodies, SAS can select the most appropriate methodologies to suit your requirements. Conjugating peptide to carrier proteins An often overlooked but critical step in generating anti-peptide antibodies is finding the best coupling strategy to cross-link peptides to an appropriate carrier molecule. Selecting the most appropriate carrier protein and choosing the correct conjugation chemistry can enormously improve the chance of producing high titre antibodies towards your antigen of interest. Peptides used for antiserum production require conjugation to a carrier since the peptide alone is usually insufficient in generating an immune response. Selection of a coupling strategy becomes extremely important, since the conjugated immunogen should present an epitope similar to that observed in the native protein, with the correct orientation and flexibility. Coupling protocols will reflect the location of the peptide sequence within native protein, amino acid composition, peptide modifications, etc. Our SAS experts work closely with clients to develop the most suitable coupling strategy. We offer peptide conjugations to standard carrier proteins such as keyhole limpet hemocyaninn (KLH), bovine serum albumin (BSA), ovalbumin (OA), among others. Contact us to discuss your specific anti-peptide antibody requirements. Labeling antibodies SAS also uses protein coupling to produce antibodies linked to various detection methods. We can cross-link antibodies for direct and indirect detection to the following labels:
Contact us to discuss the best detection methods available for your experiments. Protein cross-linking methodologies Our protein chemists are familiar with numerous cross-linking chemistries ensuring the correct conjugation protocol is used for your project. 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 composition of the immunogen must be considered since different techniques require specific chemistries. Common coupling methods will link carriers to antigens via free amino, carboxylic acid, or sulfhydryl groups. Some commonly used cross-linking reagents include:
Quality control measures Several quality control measures are available to ensure clients are supplied with the highest quality of purified proteins. We will test the purity of your antibody at several stages during purification using SDS-PAGE electrophoresis, and will quantify the increased binding affinity if an antigen is available. Additional tests can be performed upon the clients' request. Protein conjugation costs depend on the precise outcome desired, Contact us to for a quote based on your specific requirements. Order protein conjugation service from SAS Frequently asked questions - protein conjugation Q: Should my antigen be cross-linked to a carrier protein? A: 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.
Q: How do I select a carrier molecule for my peptide? A: 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.
Q: Why is my KLH-conjugated peptide solution turbid in appearance? A: 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.
Q: What technique should I use to conjugate my antigen to a carrier molecule? A: 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.
Q: Should I label my primary antibody for direct detection? A: Labeling 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.
Q: What should I use to label my antibodies? A: 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 labeled antibodies are useful in immunohistochemistry if a confocal microscope is available, and extrememly useful for immunocytochemistry and flow cytommetry.
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