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This article provides responses to the technical inquiries that we regularly receive.

How should I store my antibody?

How should I aliquot my antibody?

What concentration of primary antibody should I use?

Is the antibody validated in another species / application?

Will the antibody detect in an untested species?

Will this antibody cross react with another isoform of this protein, or protein from the same ‘family’?

What does the clone number mean?

Has this antibody been used in any publications?

How should I choose an isotype control?

How should I choose a positive control?

How should I choose a secondary antibody?

Is the information on the datasheet recent and correct?

Can you provide a free sample of a product?

Can you provide a discount?

How should I store my antibody?

Storage instructions for each antibody are provided on the datasheet. We recommend always storing the antibody as directed on the datasheet, as we are unable to guarantee how the antibody will perform if stored under different conditions.

How should I aliquot my antibody?

The volume of the aliquots will depend on how much you will typically use in an experiment. We recommend that aliquots should be no smaller than 10µl as the smaller the aliquot is, the more the concentration is affected by evaporation and adsorption of the antibody onto the surface of the vial.

What concentration of primary antibody should I use?

For antibodies that have dilution instructions for particular applications on the datasheets, we recommend following these instructions for these applications.

For antibodies that either do not have recommend dilutions on the datasheets or recommended dilutions for your particular application, we recommend using the chart below to decide a starting dilution to use based on the purity of the antibody and the application you will be using it in.

Antibody Dilution Table - Antibodies.com

Most unpurified antibodies, i.e. whole antiserum, culture supernatant or ascites fluid, will not have a concentration stated on their datasheets and these unpurified antibodies can vary significantly in specific antibody concentrations. As a rough concentration estimation, 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 dilution and concentration estimates in the chart above are recommended simply as a starting point. It may be necessary to adjust the dilution based on experimental results.

Is the antibody validated in another species / application?

All the species and applications that the manufacturer has tested the antibody in are stated on the datasheet. It is also worthwhile checking the associated product publications to learn of the species and applications that the antibody has been independently validated in.

Will the antibody detect in an untested species?

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 there are no alternative antibodies available, and you have decided it is necessary to consider purchasing an antibody for use in an untested species, we recommend checking the sequence alignment of the immunogen with the protein you are interested in.

Sequences can be found on the UniProt website, and 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, however we are still unable to guarantee how well the antibody will perform.

Will this antibody cross react with another isoform of this protein, or protein from the same ‘family’?

If cross reactivity data is available for the antibody it will be displayed on the datasheet under ‘specificity’. If this cross reactivity data is not available, we recommend checking the sequence alignment of the immunogen against the isoforms or other proteins you are interested in.

Sequences can be found on the UniProt website, and 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, therefore we recommend the alignment score should be much lower than 85% in order to indicate that there should be no cross reactivity.

What does the clone number mean?

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 number to identify it.

For more information, please read our 'Comparison of polyclonal and monoclonal antibodies’ article.

Has this antibody been used in any publications?

We list all publications that we are aware off in the ‘References’ tab on the product page. We recommend also checking CiteAb and the product pages on the manufacturers website to learn if there are any additional publications using a particular product.

How should I choose an isotype control?

Isotype controls are used to confirm that the binding of the primary antibody is specific and not a result of other protein interactions nor non-specific Fc receptor binding. The isotype control antibody should match the host species, isotype and conjugation of the primary antibody used. For example, if the primary antibody used is a HRP-conjugated rat IgG1 you will require a HRP-conjugated rat IgG1 isotype control.

How should I choose a positive control?

If you require a suitable positive control we recommend checking the datasheet first, as often it will contain a suggested positive control. It is important to ensure that the tissue or cell line used is from a tested species.

If the datasheet does not suggest a suitable positive control we recommend looking at the Uni-Prot website, as this database often has a list of tissues that the protein is expressed in. These tissues can be considered suitable positive controls. Alternatively, you can use PubMed to perform a literature search to see which cells and tissues express the protein of interest.

How should I choose a secondary antibody?

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 that you check the datasheet of the secondary antibody to ensure it has been validated in the application you will be using it.

Is the information on the datasheet recent and correct?

The datasheets contain the most up-to-date information about the products available. The tested species and applications, along with recommended dilutions, on the datasheets are always representative of the products on sale.

Can you provide a free sample of a product?

We do not offer free or trail sizes for testing purposes. Instead, our policy is that if an antibody fails to work as specified on the datasheet, we will offer a refund or replacement.

Can you provide a discount?

We do not offer discounts on individual units. However, we are able to arrange discounts on bulk purchases of five units or more. This can be five or more units of the same product, or a mixed selection of products. If you would like to place a bulk order, please contact us for further information.