Frequently Asked Questions
Common Questions and Answers about our the Services we provide
Catalogue Products
Storage instructions are provided with all antibodies. In general storage is either at +4⁰C or -20⁰C. For antibodies that are stored froze it is recommended that these are sub-aliquoted to prevent unnecessary freeze/thaw cycles. Antibodies stored at +4⁰C will generally include a preservative such as sodium azide to prevent microbial growth. This may be omitted from antibodies that are stored at -20⁰C.
Immunoglobulin concentration or required dilution range is supplied with all antibodies. It is highly recommended that the end user titrates the concentration of antibody required in their experiment for optimal results.
Custom Antibodies
We currently offer mouse and rat for monoclonal generation and rabbit for polyclonal generation.
Mycoplasmas are a range micro organisms that can infect cultures, but unlike bacterial, fungal and yeast infections, there is usually no outward signs of its presence. It can have a wide-ranging effect, altering many aspects of cellular metabolism including growth rate and protein synthesis. This can of course have a severe impact on antibody yield and performance. At FAGUS we routinely grow and test incoming hybridoma lines in isolation for mycoplasma. If a new hybridoma from a customer is found to contain mycoplasma the customer has the option to re-send an uncontaminated sample, or direct us to eliminate the mycoplasma contamination at additional cost. We will not grow and harvest antibodies from contaminated cultures due to the inevitable poor results obtained and also to prevent contamination of other customer cell lines.
FAGUS offers a range of purification methods for monoclonal and polyclonal antibodies. Typically purification starts with an affinity purification step. Purification of IgG from mouse is typically performed using Protein A derived resins, whilst Rat IgG is purified using Protein G derived resins. Protein G is also the resin of choice for polyclonal purification. These single step procedures will usually yield a purity of >= 90% with minimal losses.
For purification of IgM we suggest starting with an a series of ammonium sulphate precipitations.
For customers who require higher levels of purities we offer further steps for removal of contaminants.
• Ion Exchange Chromatography (Q-Sepharose or S-Sepharose)
• Size exclusion chromatography
• Hydroxyapatite chromatography (aggregation removal or IgM purification)
• Albumin removal (Mimetic Blue)
• Peptide resin synthesis for affinity purification of polyclonal antibodies
Products are subjected to:
SDS-PAGE
Samples are subjected to SDS PAGE under non-reducing and reducing conditions to assess intact antibody and the individual heavy and light chains. If products do not meet the customer requirement FAGUS can perform further purification using the techniques described above.
Analytical Size Exclusion Chromatography
This technique should reveal a single sharp peak corresponding to intact antibody. High molecular weight peaks that elute earlier may be due to impurities or aggregation of the antibody, whilst lower molecular weight peaks indicate impurities or degradation.
Additionally application testing can be performed.
ELISA
Antibodies can be assessed by direct or sandwich ELISA dependent upon the availability of antigens and paired antibodies.
Western Blotting
Antibodies can be tested in by western blotting against purified antigen or cell lysates dependent upon availability.
IHC
We can perform IHC using frozen or formalin fixed paraffin embedded tissue sections.
The following parameters will be reports on a Certificate of Analysis for each antibody:
• Concentration
• Amount
• Buffer
• Preservative
• Purity by SDS-PAGE
• Purity by Analytical SEC
Antibodies are supplied as frozen material and will be shipped at -20°C.
For additional materials supplied as part of a custom antibody product service, shipping conditions are as follows:
• Test bleeds will be supplied as serum and shipped frozen on dry ice
• Hybridoma cell lines will be shipped frozen on dry ice