User-supplied Antigen Requirements: For user-supplied antigens, we accept:
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recombinant proteins
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fusion proteins
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ESTs (Expressed Sequence Tags)
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peptides (conjugated or unconjugated)
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enzymes
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heat-killed bacteria and viruses, and cells
We can perform a complete custom polyclonal antibody production protocol with the following amounts of antigen:
Rabbit - New Zealand White Rabbits if the antigen is provided by the customer: 2.5-3 mg of peptide or 2.5-3 mg of protein is required. The concentration should be >0.4 mg/ml, and the antigen solution must not contain organic solvents.
Guinea pig if the antigen is provided by the customer: 2.5-3 mg of peptide or 2.5-3 mg of protein is required. The concentration should be >0.4 mg/ml, and the antigen solution must not contain organic solvents.
Chicken - Rhode Island Red the antigen is provided by the customer, 2 mg of peptide or protein is required. The concentration should be >0.4 mg/ml, and the antigen solution must not contain organic solvents.
Rat - the antigen is provided by the customer, 2.5-3 mg of peptide or protein is required. The concentration should be >0.4 mg/ml, and the antigen solution must not contain organic solvents.
Mouse - Balb/c antigens provided by the customer: 2.5-3 mg of peptide or 1.5 mg protein (>80% purity, 90% preferred) is required. The concentration should be >0.4 mg/ml, and the antigen solution must not contain organic solvents. Sheep and Goat if the antigen is provided by the customer, 5 mg of peptide or protein is required. The concentration should be >0.4 mg/ml, and the buffer must not contain organic solvents.
We can start a custom polyclonal antibody production project with 50% of the total amount of antigen needed. Additional quantities can be sent during the project’s progress. If providing a fusion protein made using a large fusion partner (such as GST or MBP), we recommend cleaving the fusion partner prior to submitting it for immunization. Large fusion partners tend to be highly antigenic, and separate purifications are needed on the resulting anti-serum to eliminate antibodies produced against these molecules. We also recommend than any antigen with a molecular weight of less than 10kDa be conjugated to a carrier protein to make it large enough to be recognized by the host animals’ immune systems. Please contact us if you are uncertain about any of the antigen requirements for custom polyclonal antibody production.
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Lyophilized Antigens: If antigens are sent lyophilized, please indicate the total amount of antigen contained, if there is a preferred storage temperature (4°C, -20°C, -80°C), and if there are any specific reconstitution steps needed.
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Antigens in Solution: If antigens are sent in solution they should be submitted at a concentration of 1mg/mL or higher (please specify the concentration, total volume, and total amount). Precipitation is not problematic for use in immunization for custom polyclonal antibody production. We need to know the buffer components that the antigen is in (PBS, Tris, < 4M urea are preferred); SDS and citrate buffers are also acceptable. It is very important that the solution not contain detergents, as these are harmful and often toxic to the animals. Please note for services such as ELISA and immuno-affinity purification the antigen needs to be soluble, and for immuno-affinity purification the protein cannot be in Tris.
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Gel Strips as Antigens: Proteins can be submitted as a gel strip, cut from an SDS PAGE gel. Please note that we cannot perform an affinity purification or ELISA when the protein is supplied in this form. The protein needs to be reasonably concentrated (2-5 mg/mL) and then run into the gel. If it precipitates, use a lower concentration. The gel is further diluted by Bio-Synthesis in 2-2.5mL of saline and 2mL of adjuvant. 100 to 200µg of protein in 500µL of gel is the maximum we can inject into rabbits. Stain the whole strip with coomassie blue (same as gel code blue stain). Then de-stain it, rinse it with water, and cut the desired strip from the gel. Do NOT “chomp” it up. After cutting the band from the gel, place it in a 15 or 50mL conical vial with an icepack – don’t freeze it. Use a fixative if desired. Please rinse twice with water to remove any remaining buffer.