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Technical frequently asked questions (FAQs)

Common technical questions, answered by our expert Technical Support Team

Questions:

  1. Can I get a free sample of a product?
  2. How should I choose a suitable secondary antibody?
  3. How should I choose an isotype control?
  4. How should I choose a positive control?
  5. How should I aliquot my antibody?
  6. How can I determine whether an antibody may detect in an untested species?
  7. Will this antibody cross react with another isoform of this protein, or protein from the same "family"?
  8. Why is the actual western blot band size different from the predicted?

1. Can I receive a free sample of a product?

We do not offer free or trial sized samples for testing purposes. Our policy is that if an antibody does not work as specified on the datasheet, we will offer a replacement or refund. If the antibody is being used in an untested species or application, we cannot offer a replacement or refund.

2.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. 

3.How should I choose an isotype control?

Isotype controls are used to confirm that the primary antibody binding is specific and not a results of non-specific Fc receptor binding or other protein interactions. The isotype control antibody should match the primary antibody host species, isotype, and conjugation.  For example, if the primary antibody is a FITC-conjugated mouse IgG1, then you will need to choose a FITC-conjugated mouse IgG1 isotype control. 

4.How should I choose a positive control?

 

You can do a quick literature search on PubMed to see which tissues and cells express the protein of interest.

5.How should I aliquot my antibody?

The size of the aliquots will depend on how much one typically uses in an experiment. Aliquots should be no smaller than 10 ; the smaller the aliquot, the more the stock concentration is affected by evaporation and adsorption of the antibody onto the surface of the storage vial.

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

CUSABIO 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. 

7.Will this antibody cross react with another isoform of this protein, or protein from the same"family"?

 We recommend checking the sequence alignment of the immunogen with the isoforms or other proteins you are interested in. There are several websites that provide a tool for calculating the percentage alignment.

Visit EMBL-EBI website

Take a copy of the immunogen sequence of the antibody and align with the protein sequence from the species you would like to test. We recommend an alignment score of over 85% as a good indication that an antibody may cross-react. We would therefore recommend that the percentage alignment should be much lower that this to indicate there should be no cross reactivity.

8.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:

  1. Post-translational modification - e.g. phosphorylation, glycosylation etc, which increases the size of the protein
  2. Post-translation cleavage - e.g. many proteins are synthesized as pro-proteins and then cleaved to give the active form, e.g. pro-caspases
  3. Splice variants - alternative splicing may create different sized proteins produced from the same gene
  4. Relative charge - the composition of amino acids (charged vs non-charged)
  5. 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

 

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