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The Quick Western Kit — IRDye® 680RD provides a universal detection reagent that can be combined with the primary antibody incubation step, eliminating the need for a secondary antibody incubation step.
This reduces the overall time to complete a Western blot and provides the advantages of near infrared fluorescence detection.
The kit also serves as a detection solution for post-immunoprecipitation samples by Western blot because it does not bind to denatured mouse monoclonal or rabbit monoclonal antibodies. (See Detection of Post-IP Proteins with Western blots using the Quick Western Kit-IRDye 680RD). The key benefit is the ability to use the same antibody for immunoprecipitation and post-immunoprecipitation detection by Western blot.
Figure 1. A431 cell lysates were immunoprecipitated overnight with a monoclonal antibody against p53. The resulting immunoprecipitates were separated by SDS-PAGE. Lane 1: Negative IP control; Lane 2: Test sample; Lane 3: A431 cell lysate positive control. Western blotting was performed using the same p53 monoclonal antibody and incubated with IRDye 680RD Immunoprecipitation Detection Reagent.
Figure 2. Two-fold dilutions of crude lysate containing an overexpressed 6X-His tagged DNA polymerase was loaded in Lanes 3-7. A Histag molecular weight marker (Invitrogen) was loaded in Lanes 1 & 9. The nitrocellulose membrane was blocked with Odyssey™ Blocking Buffer and probed with anti-His Tag Rabbit Polyclonal Antibody (GenScript; 1:500) and IRDye 680RD Detection Reagent (1:1000) for 1 hour. The image was collected on the Odyssey CLx.
The Detection Reagent has been specifically tested and qualified for Western blot applications. If additional specificity and/or affinity are required, please use IRDye secondary antibodies for detection.
IRDye 680RD Detection Reagent is known to have high specificity for IgG from:
The IRDye 680RD Detection Reagent does NOT work with chicken.