Confocal immunofluorescent analysis of mitotic HeLa cells using β-Tubulin (9F3) Rabbit mAb #2128 detected with Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) (blue) and Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb (Alexa Fluor® 555 Conjugate) #3475 (red).
Flow cytometric analysis of Jurkat cells, untreated (green) or treated with LY294002 #9901, Wortmannin #9951 and U0126 #9906 (blue), using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb #4858 detected with Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) compared to a nonspecific negative control antibody (red).
High content analysis of A549 cells exposed to varying concentrations of LY294002 (#9901) for 3 hrs, followed by 100 ng/mL EGF for 20 minutes. With increasing concentrations of LY294002, a significant decrease (~5 fold) in phospho-S6 Ribosomal Protein (Ser235/236) signal as compared to the uninhibited control was observed. When using phospho-S6 as a measurement, the IC50 of this compound was 3.06 μM. Data were generated on the Acumen® HCS platform using Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate).
Anti-rabbit IgG (H+L) F(ab')2 Fragment was conjugated to Alexa Fluor® 647 fluorescent dye under optimal conditions and formulated at 2 mg/ml. This F(ab')2 fragment product results in less non-specific binding, as it lacks the Fc domain that can bind to the cells with Fc receptors.
The optimal dilution of the anti-species antibody should be determined for each primary antibody by titration. However, a final dilution of 1:500 – 1:2000 should yield acceptable results for immunofluorescent and flow cytometry assays.
Supplied in 0.1 M sodium phosphate, 0.1 M sodium chloride, pH 7.5, 5 mM sodium azide. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.
F(ab')2 fragments are prepared from goat antibodies that have been adsorbed against pooled human serum, mouse serum, plasmacytoma/hybridoma proteins and purified human paraproteins.
This product has been optimized for use as a secondary antibody in immunofluorescent applications. Fluorescent anti-species IgG conjugates are ideal for flow cytometry and immunofluorescence. Cell Signaling Technology’s strict quality control procedures assure that each conjugate provides optimal specificity and fluorescence.
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