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Technical FAQs

  • 1. How do you make a quality antibody?

    A quality antibody is the one that has appropriate sensitivity and specificity to be able to answer the question asked by your assay. But one antibody does not necessarily work in all applications because each application has different performance specifications. Western Blots recognize denatured linearized proteins while flow cytometry, sandwich ELISA, IF, and functional assays require that antibodies should recognize the natural protein in folded shape.

  • 2. What is a clone number?

    A clone number is given to an antibody produced by a single clone of hybridoma cells and represents a specific cell line cloned from ascites that is used to produce the antibody. Each cloned cell line has a unique clone number since antibodies are produced by more than one host. The clone number is not synonymous with the lot number which is in relation with the creation of the vial. Clone refers to an individual developed from a single somatic (non-germ) cell from a parent, representing an exact replica of that parent. Also, a clone is a group of cells derived from a single ancestral cell. There is less difference amongst the quality of the clones.

  • 3. Why is the actual Western blot band size different from the predicted?

    Western blotting is a technique that separates proteins based on their size. In general, the smaller the protein the faster it migrates through the gel. However, the actual band size observed may differ from that predicted because migration is affected by common factors, including:

    a. Post-translational modification - e.g. phosphorylation, glycosylation etc., which increases the size of the protein

    b. Post-translation cleavage - e.g. many proteins are synthesized into pro-proteins and then cleaved to generate active form, e.g. pro-caspases

    c. Splice variants - alternative splicing may create different-sized proteins with the same gene

    d. Relative charge - the composition of amino acids (charged vs non-charged)

    e. Multimers - e.g. dimerisation of a protein. This is usually prevented in reducing conditions, although strong interactions can lead to the occurrence of higher bands

  • 4. What types of antigen can be used for antibody development?

    The antigens can be grouped into seven general categories: synthetic peptides (<20aa),recombinant protein fragments or full length proteins, native proteins purified from the natural source, Whole cells, DNA vaccine, small molecule Antibody.

  • 5. How can I determine whether an antibody may detect in an untested species?

    Huabio is unable to guarantee that an antibody can work in an untested species, even if the sequence alignment is high. There are many variables involved in the determination of whether or not an antibody can bind in another species. Therefore, we recommend checking the sequence alignment of the immunogen with the protein you are interested in if there are no alternatives available and it is necessary for you to consider purchasing an antibody for an untested species.

  • 6. How should I choose a suitable secondary antibody?

    Secondary antibodies should be raised against the host species to replace the primary antibody you are using. For example, you will require an anti-mouse secondary if your primary is a mouse monoclonal. We recommend you check the datasheet of the secondary antibody to ensure it is tested in the application to be used. We provide a wide range of secondary antibodies conjugated to a range of fluorochromes and chromogens.

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