Secondary Antibodies to be Relied On

Abcam boasts a full array of carefully defined secondary antibodies for which abundant validation data is available for many different important applications.

The performance of an assay depends upon the choice of a high-quality secondary antibody, since this is fundamental to ensuring the test’s specificity, sensitivity and consistency. Choosing a secondary antibody is made easier if it has already been validated in key applications, as this drastically reduces the chance of failed experiments because of undependable reagents. The challenge is the limited number of properly validated well-defined secondary antibodies.

The range of Abcam’s well-defined secondary antibodies has been shown to provide better specificity than the preadsorbed equivalents available from other suppliers, as shown in the following study. There are seven distinct secondary antibodies in Abcam’s range, which have been conjugated to HRP and biotin, thus providing complete specificity across the whole range. With each product, the datasheet contains the following information.

  • The validation data for most key applications and even for a variety of targets, in addition to comparison data from the use of competing products which serve an equivalent role, to bring out the best capabilities of key secondary antibodies
  • As a result of the high homology of immunoglobulins between species, secondary antibodies may cross-react with species other than the intended target. Pre-adsorption of the antibody against potential cross-reacting species helps to minimize this issue. Although, very few suppliers provide quantitative data on the estimated species cross-reactivity of the secondary. In the majority of cases,  suppliers do not even mention whether the secondary may potentially cross-react with other species. Each of Abcam’s datasheets contains estimated cross-reactivity for key species so you can make the best assessment possible to ensure the success of your experiment.

Unconjugated HRP Biotin
Goat anti-Rabbit IgG H&L ab182016 ab205718 ab207995
Goat anti-Mouse IgG H&L ab182017 ab205719 ab207996
Goat anti-Rat IgG H&L ab182018 ab205720 ab207997
Goat anti-Chicken IgY H&L ab182019 ab205721 ab207998
Donkey anti-Rabbit IgG H&L ab182020 ab205722 ab207999
Donkey anti-Goat IgG H&L ab182021 ab205723 ab208000
Donkey anti-Mouse IgG H&L ab182022 ab205724 ab208001

Comparative Study

Abcam’s goat anti-rabbit IgG H&L (HRP), i.e product ab205718, and two rival products, were compared using western blotting. Competitor A is an antibody which is equivalent to the above product ab205718 Competitor B is a preadsorbed antibody (to keep cross-reactivity between the antibody and non-intended species to a minimum, while keeping specificity as high as possible). As may be seen from the results, the use of ab205718 showed a significant reduction in non-target binding when compared with the preadsorbed or equivalent antibody products.

Samples Used

  • In lanes 1, 5, and 9, 10 µg of human spleen lysate were used
  • In lanes 2, 6, and 9, 10 µg of human heart lysate were used
  • In lanes 3, 7, and11, 10 µg of mouse spleen lysate were used
  • In lanes 4, 8, and 12, 10 µg of mouse heart lysate were used

Methods

The western blot was produced with a 4-12% Bis-tris gel using the MOPS buffer platform. Blotting was then transferred on to a nitrocellulose membrane before being blocked for an hour with 2% bovine serum, and then incubated with 0.05 μg/ml secondary antibodies. To identify non-specific background binding, the membrane was incubated in ECL (ab133406) and exposed for 20 minutes.

In the second study, Abcam’s goat anti-rat IgG H&L (HRP) (ab205720) was compared with another product from a competing firm which had equivalent activity. The results are shown above, and reveal that ab205720 is more specific in the detection of the primary monoclonal tubulin anti-rat antibody ab6161 in comparison to the equivalent competitor product.

Samples Used

  • Lanes 1: human liver lysate (10 μg)
  • Lanes 2: mouse liver lysate (10 μg)
  • Lanes 3: rat liver lysate (10 μg)
  • Lanes 4: HeLa whole cell lysate (10 μg)
  • Lanes 5: NIH 3T3 whole cell lysate (10 μg)
  • Lanes 6: PC12 whole cell lysate (10 μg)

Methods

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was transferred to a nitrocellulose membrane which was then blocked with 2% BSA for one hour and then incubated with ab6161 at 4 oC overnight.

The membrane was incubated in ECL (ab133406) and exposed for 15 seconds, following which the secondary antibody ab205720 (or the equivalent competing product) was then used at 1/5,000 to detect antibody binding.

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.


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Last updated: May 3, 2019 at 4:26 AM

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