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Immunofluorescent analysis of Phalloidin (green) and Calreticulin (red) in U2OS cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature and blocked with 2% BSA (Product # 37525) in PBS + 0.1% Triton X-100 for 30 minutes at room temperature. Cells were probed with a Calreticulin polyclonal antibody (Product # PA3-900) at a dilution of 1:75 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 633 goat anti-rabbit IgG secondary antibody (Product # 35562) at a dilution of 1:250 for 30 minutes at room temperature. Actin was stained with DyLight 488 Phalloidin (Product # 21833) at a dilution of 1:300 (1 unit/ml final concentration) and nuclei (blue) were stained with Hoechst (Product # 62249) at a concentration of 1ug/ml for 30 minutes. Images were taken on a Thermo Scientific ArrayScan VTI at 20X magnification.
|Tested species reactivity||Rabbit|
|Host / Isotype||Goat / IgG|
|Purification||Antigen affinity chromatography|
|Storage buffer||PBS, pH 7.4, with 1% BSA|
|Contains||0.02% sodium azide|
|Storage Conditions||4° C|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:25 - 1:100|
|Immunocytochemistry (ICC)||1:50 - 1:2,000|
|Immunofluorescence (IF)||1:50 - 1:2,000|
|Immunohistochemistry (IHC)||1:50 - 1:2,000|
|Immunoprecipitation (IP)||Assay Dependent|
|Western Blot (WB)||1:5,000 - 1:20,000|
* 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.
Product # 35562 has been successfully used in Western blot, IF, ICC, IHC, IP and FACS applications.
Product # 35562 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. 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. DyLight 633 Amax= 638 nm; Emax= 658 nm. Mole Dye/Mole Protein Ratio is lot-dependent.
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.