FAQ: Antibodies & Proteins

How should I choose a suitable secondary antibody?

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

How should I choose an isotype control?
Isotype controls are used to confirm that the primary antibody binding is specific and not a result of non-specific Fc receptor binding or other protein interactions. The isotype control antibody should match the primary antibody’s 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. Most isotype controls are monoclonals. They are not suitable for use with polyclonal antibodies as these contain more than one IgG subclass.

What concentration of primary antibody should I use?
On the product datasheet we indicate recommended dilutions. However, these dilutions are only recommended as a starting point and it may be necessary to adjust the dilution based on the experimental results.

How should I store the antibody?
We recommend always storing the antibody as described on the product datasheet. We do not guarantee the antibody performance if not stored as stated on the product datasheet.

How should I aliquot the antibody?
The size of the aliquots depends on how much one typically uses in an experiment. However, aliquots should be no smaller than 10 µl. 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.

Are there any publications using this antibody?
All publications that we are aware of using a particular antibody will be listed on the "Literature References" tab of the product datasheet. If we find additional information, the product datasheets are updated live on the website immediately.

Is the antibody tested in another species / application?
All the species and applications of the antibody have been tested as stated on the product datasheet. If you do not find a specific application or species listed on our product datasheet and if AdipoGen has no further in-house data, Technical Support can check their reference database to see if other researchers have published this use, sample type, and/or species with this antibody.

If you decide to use a product in an untested application or species, we encourage you to send us your results, whether positive or negative. Such information is made available to other researchers immediately.

Will this antibody cross-react with another isoform of this protein?
For some of the antibodies cross-reactivity data is available. Please check the ‘cross-reactivity’ section on the product datasheet. If no information is indicated, we recommend checking the sequence alignment of the immunogen with the isoforms or other proteins in which you are interested in.

Can AdipoGen provide the immunogen sequence for an antibody?
For some of the antibodies the immunogen sequence is not readily available on the product datasheet. If you require such information for your experiments please contact our Technical Support.

Why is (in some cases) 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. The observed band size may differ from that predicted due to:

1. Post-translational modifications - e.g. phosphorylation, glycosylation, etc. which increase the size of the protein.
2. Post-translation cleavage - many proteins are synthesized as pro-proteins and then cleaved to give the active form, e.g. pro-caspases, pro-interleukins, etc.
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.

Which epitope does the monoclonal antibody recognize?
AdipoGen does generally not epitope map its antibodies. The antibody is usually generated using the entire immunogen as listed on the product datasheet. If a peptide immunogen is used, it will be indicated on the product datasheet.

Which epitope does the polyclonal antibody recognize?
Polyclonal antibodies generally have multiple epitope recognition. The antibody is usually generated using the entire immunogen as listed on the product datasheet. If a peptide immunogen is used, it will be indicated on the product datasheet.

What is the expiration date of AdipoGen proteins and antibodies?
All products supplied by AdipoGen are warranted to meet or exceed the published specifications when stored under the recommended proper storage conditions, for at least 1 year upon delivery. The antibody performance cannot be warranted after the expiration date. However, under proper storage conditions, proteins and antibodies tend to be stable for many years. These conditions include storing proteins as lyophilized powder, storing the product frozen (-20°C or -80°C) at protein concentrations of greater than 0.1 mg/mL, and limiting the number of freeze/thaw cycles. Please review the individual product inserts for specific instructions.

How do I convert kDa to grams/mol?
On the product datasheets AdipoGen lists the molecular weight of the proteins in kDa. The approximate conversion to molar mass is 1 dalton=1 gram/mol (i.e. a 20 kDa protein is equal to 20,000 grams/mol).

What are AdipoGen‘s Matched Pair Detection Pack?
AdipoGen‘s Matched Pair Detection Pack are a do-it-yourself immunoassay type products, which contain a set of main reagents that allow to assemble a specific kit (no plate or buffers provided). The Pack is provided with a comprehensive manual.

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