F(ab')2-Goat anti-Rabbit IgG (H+L) Cross-Adsorbed, Alexa Fluor™ 647 (A-21246) (2024)

Multiplexed fluorescent western blot was performed using F(ab)2-Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (Product # A-21246). Whole cell extracts of THP-1 (Lane 1, 2, 3, 4), MCF7 (Lane 5, 6, 7), HeLa (Lane 8), and HEK 293 (Lane 9) were electrophoresed usingNuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were transferred onto anitrocellulose me... View More {{ $ctrl.currentElement.advancedVerification.fullName }} validation info. View more

• Alexa Fluor 350
• Alexa Fluor 488
• Alexa Fluor 546
• Alexa Fluor 555
• Alexa Fluor 680
• Alexa Fluor 790
• Alexa Fluor Plus 488
• Alexa Fluor Plus 555
• Alexa Fluor Plus 647
• AP
• Biotin
• Cyanine3
• Cyanine5
• FITC
• HRP
• PE
• Rhodamine
• Rhodamine Red-X
• Texas Red
• Texas Red-X

To minimize cross-reactivity, these goat anti-rabbit IgG (H+L) divalent F(ab')2 secondary antibodies have been affinity purified and cross-adsorbed against pooled human serum, mouse serum, mouse plasmacytoma/hybridoma proteins, and purified human paraproteins. Cross-adsorption or pre-adsorption is a purification step to increase specificity of the antibody resulting in higher sensitivity and less background staining. The secondary antibody solution is passed through a column matrix containing immobilized serum proteins from potentially cross-reactive species. Only the nonspecific-binding secondary antibodies are captured in the column, and the highly specific secondaries flow through. The benefits of this extra step are apparent in multiplexing/multicolor-staining experiments (e.g., flow cytometry) where there is potential cross-reactivity with other primary antibodies or in tissue/cell fluorescent staining experiments where there are may be the presence of endogenous immunoglobulins.

Alexa Fluor dyes are among the most trusted fluorescent dyes available today. Invitrogen™ Alexa Fluor 647 dye is a near-infrared-fluorescent dye with excitation ideally suited to the 647 nm laser line. For stable signal generation in imaging and flow cytometry, Alexa Fluor 647 dye is pH-insensitive over a wide molar range. Probes with high fluorescence quantum yield and high photostability allow detection of low-abundance biological structures with great sensitivity. Alexa Fluor 647 dye molecules can be attached to proteins at high molar ratios without significant self-quenching, enabling brighter conjugates and more sensitive detection. The degree of labeling for each conjugate is typically 2-8 fluorophore molecules per IgG molecule; the exact degree of labeling is indicated on the certificate of analysis for each product lot.

Using conjugate solutions: Centrifuge the protein conjugate solution briefly in a microcentrifuge before use; add only the supernatant to the experiment. This step will help eliminate any protein aggregates that may have formed during storage, thereby reducing nonspecific background staining. Because staining protocols vary with application, the appropriate dilution of antibody should be determined empirically. For the fluorophore-labeled antibodies a final concentration of 1-10 µg/mL should be satisfactory for most immunohistochemistry and flow cytometry applications.

Product will be shipped at Room Temperature.