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Technical FAQs

  • 1.How do you make a quality antibody?

    A quality antibody is one that has the appropriate sensitivity and specificity to be able to answer the question asked by your assay. Also, one antibody will 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 antibodies that recognize the natural, folded shape of the protein.

  • 2.What is a clone number?

    A clone number is given to an antibody produced by a single clone of hybridoma cells. Each clone number represents a specific cell line cloned from ascites that was used to manufacture the antibody. Since antibodies are produced by more than one host, each cloned cell line receives a unique clone number. The clone number is not synonymous with the lot number which is in relation with the creation of the vial. There is little to no difference amongst the quality of the clones.Clone also refers to an individual developed from a single somatic (non-germ) cell from a parent, representing an exact replica of that parent. A clone is a group of cells derived from a single ancestral cell.

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

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

    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 as pro-proteins and then cleaved to give the active form, e.g. pro-caspases

    c. Splice variants - alternative splicing may create different sized proteins produced from 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 result in the appearance 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 are unable to guarantee that an antibody will work in an untested species, even if the sequence alignment is high. There are many variables involved in the determining whether an antibody will bind in another species. If there are no alternatives available, and it is necessary for you to consider purchasing an antibody for use in a species that is not tested, we recommend checking the sequence alignment of the immunogen with the protein you are interested in.

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

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