Immunofluorescence analysis of Goat anti-Rabbit IgG (H+L) Cross Adsorbed Secondary Antibody, Rhodamine conjugate was performed using HeLa cells stained with alpha Tubulin Rabbit Polyclonal Primary Antibody (PA516891). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, blocked with 1% BSA for 1 hour and labeled with 2ug/ml Rabbit primary antibody for 3 hours at room temperature. Goat anti-Rabbit IgG (H+L) Cross Adsorbed Secondary Antibody, Rhodamine conjugate (31686) was used at a concentration of 4ug/ml in phosphate buffered saline containing 0.2% BSA for 45 minutes at room temperature, for detection of Alpha tubulin in the cytoplasm (Panel a: red). Nuclei (Panel b: blue) were stained with DAPI in SlowFade® Gold Antifade Mountant (S36938). F-actin was stained with Alexa Fluor® 488 Phalloidin (A12379, 1:300) (Panel c: green). Panel d represents the composite image. No nonspecific staining was observed with the secondary antibody alone (panel f), or with an isotype control (panel e). The images were captured at 60X magnification.
|Tested species reactivity||Rabbit|
|Host / Isotype||Goat / IgG|
|Storage buffer||PBS, pH 7.6, with 15mg/ml BSA|
|Contains||0.05% sodium azide|
|Storage Conditions||4° C|
|Cross Adsorption||Against human, goat, mouse and sheep serum proteins|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:50 - 1:200|
|Immunocytochemistry (ICC)||4 µg/ml|
|Immunofluorescence (IF)||4 µg/ml|
|Immunohistochemistry (IHC)||1:50 - 1:200|
|Immunoprecipitation (IP)||1:50 - 1:200|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Concentration may vary slightly from lot-to-lot, see lot-specific datasheet for exact concentration.
Product # 31686 has been successfully used in Western blot, IF, ICC, IHC, IP and FACS applications.
Product # 31686 reacts with the heavy chains of rabbit IgG and with the light chains common to most rabbit immunoglobulins, but does not react against non-immunoglobulin serum proteins. This antibody has been tested by ELISA and/or solid-phase adsorbed to ensure minimal cross-reaction with human, goat, mouse, and sheep serum proteins. However, this antibody may cross-react with immunoglobulins from other species.
Store product protected from light at 4°C until opened. To extend the shelf-life of this product, add an equal volume of glycerol to make a final concentration of approximately 50% glycerol and store at -20°C. Rhodamine Amax= 550 nm; Emax= 570 nm. Fluorophore/Protein: 0.5 moles Rhodamine per mole IgG. (lot-dependent).
Reconstitute with 1.1 ml of distilled water (1.5 mg/ml after restoration).
Country of Origin: USA
Thermo Scientific Anti-Rabbit secondary antibodies are affinity-purified antibodies with well-characterized specificity for rabbit immunoglobulins and are useful in the detection, sorting or purification of its specified target. Secondary antibodies offer increased versatility enabling users to use many detection systems (e.g. HRP, AP, fluorescence). They can also provide greater sensitivity through signal amplification as multiple secondary antibodies can bind to a single primary antibody. Most commonly, secondary antibodies are generated by immunizing the host animal with a pooled population of immunoglobulins from the target species and can be further purified and modified (i.e. immunoaffinity chromatography, antibody fragmentation, label conjugation, etc.) to generate highly specific reagents.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.