We recommend always storing the antibody as instructed on the datasheet. We are unable to guarantee how the antibody will perform if it is stored under different conditions.
The size of the aliquots will depend on how much one typically uses in an experiment. The aliquots should be no smaller than 10µl. The smaller the aliquot is, the more the concentration is affected by evaporation and adsorption of the antibody onto the surface of the vial.
Many of our antibodies have dilution instructions on their datasheets. In these instances, we recommend following these instructions. For antibodies that do not have recommend dilutions, we recommend using the chart below to decide a starting dilution.
| Tissue Culture Supernatant | Ascites | Whole Antiserum | Purified Antibody | |
|---|---|---|---|---|
| WB / DB | 1/100 | 1/1,000 | 1/500 | 1 µg/ml |
| FC | 1/100 | 1/1,000 | 1/500 | 1 µg/ml |
| ELISA | 1/1,000 | 1/10,000 | 1/500 | 0.1 µg/ml |
| IHC / ICC | Neat - 1/10 | 1/100 | 1/50 - 1/100 | 5 µg/ml |
| IP | --- | 1/100 | 1/50 - 1/100 | 1-10 µg/ml |
Most unpurified antibodies (i.e. whole antiserum, culture supernatant, or ascites fluid) will not have a concentration stated on their datasheets - as it will not have been determined. These antibodies can vary significantly in specific antibody concentrations. As a rough concentration estimate, tissue culture supernatant is 1-3 mg/ml, ascites is 5-10 mg/ml, and whole antiserum is 1-10 mg/ml.
It is important to remember that the dilutions / concentrations in the chart above are recommended simply as a starting point. It may be necessary to adjust the dilution / concentration based on experimental results.
We list all the species and applications that we are aware of an antibody being validated in on the datasheet.
We are unable to guarantee that an antibody will work in an untested species, even if the sequence alignment is high, as there are many variables involved in determining whether an antibody will bind in another species.
If cross reactivity data is available for the antibody, it will be displayed on the datasheet under the "specificity" and "cross-reactivity" headings. If cross reactivity data is not available, we recommend checking the sequence alignment of the immunogen with the isoform(s) or other protein(s) you are interested in.
An online tool for calculating the percentage alignment can be found on the EMBL-EBI website. You will need to take a copy of the immunogen sequence of the antibody and align it with the protein sequence from the species you would like to test. We recommend an alignment score of > 85% as a good indication that an antibody may cross-react. We recommend the alignment score should be much lower than 85% in order to indicate that there should be no cross reactivity.
Each clone number represents a specific cell line cloned from ascites that was used to manufacture the antibody. Since monoclonal antibodies are produced by more than one host and more than one cell line, each cloned cell line receives a unique clone number to identify it.
We list all publications that we are aware of in the ‘Citations’ tabs. We recommend also checking CiteAb to see if there are any additional citations for a particular product.
Isotype controls are used to confirm that the binding of the primary antibody is specific and not a result of other protein interactions or non-specific Fc receptor binding.
The isotype control antibody should match the primary antibodies host species, isotype, and conjugation. For example, if the primary antibody is HRP-conjugated rat IgG1, then you will need a HRP-conjugated rat IgG1 isotype control.
We recommend checking the datasheet first, which will often have a suggested positive control. It is important to ensure that the tissue or cell line used is from a tested species.
If no positive controls are suggested, we recommend looking at the UniProt website. This database often has a list of tissues that the protein is expressed in and these tissues can be considered suitable positive controls.
Secondary antibodies should be raised against the host species of the primary antibody you are using. For example, if your primary antibody is a rat monoclonal you will require an anti-rat secondary antibody. We recommend checking the datasheet of the secondary antibody to ensure that it has been validated in the application you will be using.
The datasheets contain the most up-to-date information available about a product. When we find additional information, the datasheets are updated immediately, so they should never be out-of-date.