Western blot analysis was performed on membrane-enriched extracts (30 µg lysate) of K-562 (Lane 1) and U-87 MG (Lane 2). The blots were probed with ABfinity™ Anti-PRDX6 Rabbit Monoclonal Antibody (Product # 702211, 2 µg/ml) and detected using Donkey anti-Rabbit IgG Secondary Antibody, WesternDot 800 (Product # W10824) at dilutions 1:1,000 (Fig. 1), 1:2,500 (Fig. 2) and 1:5,000 (Fig. 3). A 22 kDa band corresponding to SOD2 was observed across the cell lines tested. PRDX6 was multiplexed with Anti-beta-3 Tubulin Mouse Monoclonal antibody (Product # 32-2600, 1:4000 dilution) and detected using Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 680 (Product # A-21057, 0.2 µg/ml). A 52 kDa band corresponding to beta-3 Tubulin was observed. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary antibodies after blocking with 5 % skimmed milk. Fluorescent detection was performed using the Odyssey® Fc imaging system (Li-cor Biosciences).
|Tested species reactivity||Rabbit|
|Host / Isotype||Donkey / IgG|
|Immunogen||Rabbit Secondary Antibody|
|Storage buffer||50mM borate, pH 8.3, with 1M betaine|
|Contains||0.05% sodium azide|
|Storage Conditions||4° C, do not freeze|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Western Blot (WB)||1:1,000-1:5,000|
* 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.
WesternDot fluorescently labeled secondary antibodies deliver superior signal-to-noise ratios and exhibit comparable sensitivity to enhanced chemiluminescence (ECL)-based methods in western blot applications. No stripping and re-probing or multiple blots are required for multiple protein detection as commonly needed with ECL or colorimetric detection. Differently labeled WesternDot antibodies can be applied to a single blot for simultaneous detection of multiple proteins using standard gel or blot imaging platforms (a list of instruments that are compatible with WesternDot detection can be found in the user manual below). Our WesternDot antibodies utilize Qdot nanocrystals enhanced with VIVID technology, making them brighter than the original Qdot reagents. Additionally, they have reduced intensity differences between the different colors, simplifying multiplex applications.
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.