In practical terms, the Nano-Secondary is mixed with the primary antibody and the primary antibody’s protocol is used for Western blotting.įigure 6: Multiplex fluorescent Western blot of GFP-TOM70, ß-Tubulin, and GFP in HEK293T cell lysate: Membrane was simultaneously incubated with primary antibodies and Nano-Secondary reagents. This way, it is not necessary to strip and re-probe the Western blot membrane. This allows multiple targets to be analyzed simultaneously on the same blot at the same time. Yes, multiple Nano-Secondary reagents can be applied in parallel for multiplex fluorescent Western blotting. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.ĭoes multiplex fluorescent Western blotting work with Nano-Secondary reagents?
#Purpose of secondary antibody in western blot professional
Confocal images were acquired with a Leica TCS SP8 microscope, 100x oil objective, and deconvolved with Huygens Professional (SVI). Magenta: mouse IgG2b anti-Tubulin + alpaca anti-mouse IgG2b V HH Alexa Fluor 647. Green: mouse IgG1 anti-COX4 + alpaca anti-mouse IgG1 V HH Alexa Fluor 488. Yellow: rabbit anti-Lamin + alpaca anti-rabbit IgG V HH Alexa Fluor 568. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.įigure 5: Multiplexing of HeLa cells with alpaca anti-rabbit and anti-mouse Nano-Secondary reagents. One-step or parallel incubation of primary antibody and corresponding Nano-Secondary reagent is used for immunofluorescence, super-resolution microscopy, Western blotting, and flow cytometry.įigure 4: Multiplexing immunofluorescence a using triple mouse secondary antibody staining: 3 subclass specific anti-mouse Nano-Secondary reagents: Immunofluorescence/ confocal microscopy of HeLa cells with alpaca anti-rabbit Nano-Secondary reagents : From left to right Mouse IgG2b anti-Lamin + alpaca anti-mouse IgG2b V HH Alexa Fluor 488 Mouse IgG3 anti-MOT + alpaca anti-mouse IgG3 V HH Alexa Fluor 568 Mouse IgG1 anti-Vimentin + alpaca anti-mouse IgG1 V HH Alexa Fluor 647. Yes, please find the protocol here: O ne-step immunostaining for immunofluorescenceįor what applications can I use Nano-Secondary reagents? Is there a protocol for one-step immunostaining for immunofluorescence? In practical terms, the Nano-Secondary reagent is added to the primary antibody and the primary antibody’s protocol is used for immunostaining.įigure 3: Experimental procedure of one-step immunostaining This results in a one-step immunostaining. Epifluorescence images were acquired with CellInsight CX7 HCS microscope, 20X objective.īecause the Secondary reagents are monovalent and bind with high specificity and affinity to their target IgGs, they can be simultaneously incubated with the primary antibody. The simultaneous incubation of primary antibody and Nano-Secondary reagent also supports multiplexing, live-cell immunostaining, and improves cell viability for flow cytometric analysis.įigure 2: Comparison with classical sequential staining of primary antibody and Nano-Secondary reagent: HeLa cells were immunostained with rabbit anti-βActin and anti-LaminB1 and secondary anti-rabbit IgG V HH Alexa Fluor647. This method reduces incubation time, hands-on time and the number of washing steps. One-step immunostaining is the simultaneous incubation of primary antibody and Nano-Secondary reagent.
Currently, Nano-Secondary reagents that bind to anti-rabbit or subclass-specific anti-mouse IgGs are available.įigure 1: Nano-Secondary reagents are alpaca Nanobodies conjugated to fluorescent dyes that specifically bind to primary antibodies. Nano-Secondary reagents are conjugated to Alexa Fluor ® dyes. Nano-Secondary reagents consist of alpaca Nanobodies/ V HHs that bind to primary antibodies with high affinity in a species and subclass-specific manner. ChromoTek’s Nano-Secondary reagents are novel secondary antibodies for higher resolution, cleaner images, and faster immunostaining.
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