Antibody Technologies FAQ

Find answers to the most commonly asked questions regarding our antibodies, applications, and protocols. 

Questions on Primary Antibodies

We are the manufacturer and supplier of two brands of primary antibodies, Triple A Polyclonals and PrecisA Monoclonals.

Triple A Polyclonals are advanced research-grade rabbit polyclonals, originally developed as part of the Human Protein Atlas project to create a protein expression map of the complete human proteome. You can find over 21,000 Triple A Polyclonals in our catalog, validated for IHC, ICC and Western blot.

PrecisA Monoclonals are mouse monoclonal antibodies. The development of the PrecisA Monoclonals is based on the knowledge from the Human Protein Atlas with careful antigen design and extended validation of antibody performance. Each PrecisA Monoclonal is epitope mapped and provided with the most relevant characterization data for its specific target.

 

Application specific questions

In addition to selected IHC stainings available on the product data pages, you can access all IHC images from human tissues on the Human Protein Atlas (HPA) portal. To find this information, follow the link under the “Additional Images” on the product page of the antibody you are interested in. The high-resolution IHC images can be found on the Human Protein Atlas portal by clicking on the different tissues. The IHC images from cancer tissues are found on the Pathology Atlas.  

All IHC images are annotated manually by skilled professionals. To achieve an unbiased annotation of each antibody, based on the staining pattern alone, the first step is performed without consulting any literature. In the next step, another team of researchers compares the IHC staining results for each antibody with available literature. The staining result of each antibody is also compared to RNA sequencing data from 27 different human cell lines, as well as with the results using other antibodies against the same target, when available.

As an additional layer of validation, enhanced validation is performed in IHC using orthogonal methods and validation by independent antibodies. Learn more about our validation here and the different enhanced validation methods here.

Our standard antigen retrieval method is Heat Induced Epitope Retrieval (HIER) in retrieval buffer pH 6. Please refer to our IHC protocol for more information. 

During IHC validation, the antibodies are evaluated on paraffin-embedded tissues and cells, however, our experience is that they usually perform well in fresh frozen tissues as well.

We recommend the following protocols for using our antibodies in IHC:

You can find all our protocols here

A large number of our antibodies are approved for Western Blot. The antibodies are analyzed on endogenous human cell lines, tissue protein lysates, or recombinant full-length human protein lysates. Learn more about how our antibodies are validated in Western blot here.

There are several reasons why an actual Western Blot (WB) band size may differ from a predicted size. Such reasons include: 

  • Splice variants: one gene can generate proteins of different sizes due to alternative splicing.  
  • Post-translational modifications like glycosylations and phosphorylations increase the molecular weight of the target proteins.   
  • Post-translational cleavage to generate the active form of pro-proteins results in a change of the molecular weight, e.g. proinsulin and insulin.
  • Dimers and multimers can be present even though the sample run under reducing conditions.
  • Hydrophobic proteins, such as transmembrane proteins, may have difficulties in migrating through the gel resulting in different multi-bands patterns. 

All cell and tissue lysates used for Western Blot are prepared using the Proteoextract Complete Mammalian Proteome Extraction Kit (Calbiochem).

Here is the standard protocol we recommend for Western blot using our products. 

If your antibody requires BSA blocking, please use this alternative Western blot protocol using BSA blocking instead.

You can find all our protocols here. 

A large number of our antibodies are recommended for immunofluorescence in cell lines / Immunocytochemistry (ICC). The product page for each antibody contains information on all applications and species the antibody is validated for.

Besides, for ICC-validated Triple A Polyclonals, there is a link to the Cell Atlas (ICC-IF characterization data) in the Human Protein Atlas database showing additional images from several cell lines. In the Cell Atlas, it is possible to choose how to visualize the ICC-IF images from the selected cell lines with one or several reference markers by switching on/off the following channels: green (for the antibody staining); blue (for the nucleus); red (for the microtubules), and yellow (for the endoplasmic reticulum, ER).

The ICC-IF protocol we recommend for using with our antibodies is available here. 

You can find all our protocols here.

Our antibodies are tested for recognition of its antigen (recombinant protein) in an ELISA-setup. However, ELISA is currently not one of our validated applications. If you are interested in testing one of our antibodies in ELISA, you are welcome to join our Explorer Program. 

The product page specifies in which applications and species each antibody are currently validated for. If the species or the application you are interested in is not listed, please contact our Scientific Support

If you are interested in testing one of our antibodies in a currently not supported species or application, you are welcome to join our Explorer Program.

Questions on The Human Protein Atlas

The Human Protein Atlas is a Swedish academic project aiming at characterizing the complete human proteome using antibodies. The Human Protein Atlas database offers a map of the human protein expression and is a valuable tool for researchers. 

The antibodies developed within the project are manufactured and distributed by Atlas Antibodies under the brand name Triple A Polyclonals. The characterization data for each antibody is freely accessible online.

Questions on validation of antibodies

Triple A Polyclonals and PrecisA Monoclonals are carefully designed using a proprietary antigen design software which selects a Protein Epitope Signature Tag (PrEST) sequence of between 50 to 150 amino acids having the lowest possible identity to other human proteins.  The PrEST concept secures the highest level of specificity when used for antibody production. 

Triple A Polyclonals are derived from rabbits and PrecisA Monoclonals from mice.

The antibodies are developed against human protein fragments and are validated against human tissues and cells. A selection of the antibodies is also validated in rodent samples. The antibodies pass through a series of validation steps, including specific target recognition on protein array (or ELISA), testing using IHC, WB, and ICC-IF. Achieved results are compared to literature, to RNA sequencing information, and to the results by other antibodies against the same target, when available. A description of our validation process in the different applications is available on the Antibody Validation page. In addition to standard validation, enhanced validation is performed using five validation methods: genetic validation, orthogonal validation, validation by independent antibodies, recombinant expression validation and migration capture MS validation.

Our antibodies are validated for one or more applications such as IHC, WB and ICC. All the information about the applications an antibody has been validated for is available in the antibody product page.

If you are interested in testing our antibodies in an application or species not yet recommended for a certain antibody, you are welcome to join our Explorer Program.  In this program, we offer a 50% discount on the next vial you purchase or alternatively, a 50% refund, as soon as you share your results with us. 

Many of our antibodies recognize the corresponding mouse and rat proteins as well. This information is reported on each antibody’s product page. Information about antigen sequence identity to mouse and rat orthologs is reported on the product datasheets for all antibodies. 

If you are interested in testing our antibodies in a species or application not yet recommended for a certain antibody, you are welcome to join our Explorer Program. In this program, we offer a 50% discount on the next vial you purchase or, alternatively, a 50% refund, as soon as you share your results with us.

General questions

You can find all our recommended protocols here

Please see the product page for the antibody you are interested in to find recommended dilutions for each application.

Triple A Polyclonals are produced using a standardized production process to ensure consistency. By a unique three-step antibody purification process, using the recombinant PrEST-antigen as the affinity ligand, only target specific antibodies are collected.

PrecisA Monoclonals are protein A purified.

No, currently all our antibodies are non-conjugated.

If we have more than one PrecisA Monoclonal with the same product name, it means that the antibodies either have different isotypes or recognize different epitopes. When these antibodies show the same or a similar staining pattern, this enhances the reliability of the antibodies. This is of great importance in particular for previously uncharacterized proteins, where literature describing tissue and subcellular location of the target proteins is very limited.

To choose which product would work best for your needs please refer to the product datasheets. If you want assistance we are happy to help.

We are proud to have an increasing number of our products referenced in articles published in high-impact journals. The references for our products are regularly updated on the product page of each product.

If you have used one of our products (antibodies, PrEST Antigens or QPrESTs) in one of your publication, please contact us at support@atlasantibodies.com.

If we have two different Triple A Polyclonals with the same product name, it means that the antibodies are directed against different regions of the same protein. When these antibodies show the same or a similar staining pattern, this enhances the reliability of the antibody itself. This is of great importance in particular for previously uncharacterized proteins, where literature describing tissue and subcellular location of the target proteins is very limited.

To choose which product would work best for your needs please refer to the product datasheets. If you want assistance we are happy to help.

The design of our antibodies is based on the information stated in the ENSEMBL database. On the product page of each antibody, there is a link to the corresponding antibody page on the Human Protein Atlas portal. There you can find the identity of the antigen sequence to matching protein-coding ENSEMBL gene transcripts. In addition, at the bottom of the page, there is a feature named Antigen View. This graphical view allows you to study the position of the antigen sequence on the target protein compared to matching transcripts or isoforms. The antigen sequence is visualized as a horizontal green bar. You are always welcome to contact our Scientific Support if you need help.

Yes, we do have a Guarantee Program which applies to all our products. On this page, you can read more about it.

We do not provide small test samples at this time. We have a Guarantee Program where we offer a free vial or your money back if the antibody does not perform as expected in validated applications and an Explorer Program for testing antibodies in not yet validated applications or species. All our IHC recommended antibodies are extensively characterized on the Human Protein Atlas website and the staining patterns achieved using the antibodies in the majority of human tissues can be carefully investigated before purchase.

Questions on PrEST Antigens

PrEST Antigens are the immunogens used for the generation of Triple A Polyclonals and PrecisA Monoclonals. The PrEST Antigens are useful as control antigens, for example as blocking agents and positive assay controls together with the corresponding antibody. They are recombinant human Protein Epitope Signature Tags (PrESTs) of approximately 50 to 150 amino acids expressed as fusion proteins with a dual tag consisting of His6 and Albumin Binding Protein (ABP). The protein-specific PrEST sequences are specified on the product pages for all antibodies.

 

The PrEST Antigens are designed to have a sequence identity as low as possible to other human proteins. The selection of PrESTs is based on a sliding window algorithm to predict the local and regional sequence identity of the various parts of a particular human protein to all the other protein sequences of the human proteome. Signal peptides and membrane regions are avoided using different software including Phobius, SPOCTOPUS, and SignalP.

The dual tag consists of a hexahistidyl (His6) tag in frame with an immunopotentiating Albumin Binding Protein (ABP)-tag. The His6 tag can be used for IMAC-purification and proteins fused to ABP, originating from the albumin binding region of Streptococcal Protein G, can be purified by Human Serum Albumin (HSA) affinity chromatography.

The sequence of the dual tag is: GSSHHHHHHSSGLVPRGSHMASLAEAKVLANRELDKYGVSDYHKNLINNAKTVEGVKDLQAQVVESAKKARISEATDGLSDFLKSQTPAEDTVKSIELAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPGTFAHYMDPNSSSVDKLAAA

The specific human protein sequence begins directly after “VDKLAAA”.

 

The PrEST Antigens are purified using nickel-containing matrices (IMAC) and the purity is analyzed using SDS page.

For cloning of the PrESTs, a pool of RNA consisting of material from several human tissues is used in an RT-PCR approach. The amplified gene fragments are cloned into the expression vector pAff8c using solid-phase cloning with biotinylated primers and magnetic beads and all clones are sequences verified. An E. coli (Rosetta) recombinant protein expression system is used for expressing the clones.

Some PrEST Antigens require 1 M Urea to be soluble. If you want to use a lower Urea concentration, we advise you to first try this with a small aliquot of your sample.

Contact Scientific Support

Need help with finding the right product or an advice on applications? Contact our Scientific Support.

Contact Support