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In order to achieve high assay performance, selection of a good quality secondary antibody is critical to ensure specificity, sensitivity and consistency. When it comes to choosing a secondary, validation in key applications minimizes the risk of experimental failure due to unreliable reagents. Unfortunately, the availability of well-defined secondary antibodies with validation data is limited.
Our range of well-defined secondaries have proved more specific than both equivalent and preadsorbed products from competitors (see comparative study below). We have developed seven different secondary antibodies and conjugated them to HRP and biotin, ensuring high specificity across the entire range. Each product datasheet contains:
|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|
The performance of our Goat anti-Rabbit IgG H&L (HRP) ( ab205718) was compared by western blotting to two competitor products. Competitor A is equivalent to ab205718, while competitor B has been pre-adsorbed to minimize species cross-reactivity and maximize specificity. The results show that non-specific binding is significantly decreased when using ab205718 in comparison to equivalent and preadsorbed products from competitors.
Lanes 1, 5, and 9: Human spleen lysates (10 μg)
Lanes 2, 6, and 10: Human heart lysates (10 μg)
Lanes 3, 7, and 11: Mouse spleen lysates (10 μg)
Lanes 4, 8, and 12: Mouse heart lysates (10 μg)
This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was transferred onto a nitrocellulose membrane and then blocked for an hour using 2% Bovine Serum Albumin before being incubated with secondary antibodies at 0.05 μg/ml. Any non-specific background binding was detected following membrane incubation in ECL (ab133406) and 20 minute exposure.
The performance of our Goat anti-Rat IgG H&L (HRP) (ab205720) was compared by western blotting to an equivalent competitor product. The results show the higher specificty of ab205720 in comparison to the equivalent product from a competitor when detecting primary antibody ab6161 (Rat monoclonal to tubulin).
Lanes 1: Liver (Human) tissue lysate (10 μg)
Lanes 2: Liver (Mouse) tissue lysate (10 μg)
Lanes 3: Liver (Rat) tissue 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)
This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was transferred onto a nitrocellulose membrane and then blocked for an hour using 2% BSA before being incubated with ab6161 overnight at 4C. Antibody binding was detected using ab205720 secondary antibody or a equivalent product from a competitor at 1/5,000 following membrane incubation in ECL (ab133406) and 15 seconds exposure.