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Quick Western Kit - IRDye® 680RD

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.

A431 cell lysates were immunoprecipitated overnight with a monoclonal antibody against p53.

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.

Two-fold dilutions of crude lysate containing an overexpressed 6X-His tagged DNA polymerase was loaded in Lanes 3-7.

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.

Saves Time (View Workflow)

  • Does not require a separate primary antibody labeling step, saving time and antibody
  • Faster method of detection compared to the traditional 2-step method, which can take up to 4 hours
  • Reduces the total Western blot procedure by at least 90 minutes

Convenient

  • Eliminates the use of labeled secondary antibodies and associated washes
  • Eliminates the need to purchase multiple secondary antibodies
  • Use the same antibody for immunoprecipitation (IP) and post-IP detection by Western blot

Universal Detection

  • Works with a variety of primary antibodies (see list below)
  • Has been shown to recognize primary antibodies to recombinant tagged proteins (i.e. 6X His, Myc, FLAG, etc.)

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:

  • Human
  • Mouse
  • Rabbit
  • Guinea Pig
  • Goat
  • Sheep
  • Pig
  • Cow
  • Cat
  • Dog
  • Donkey
  • Chicken

    The IRDye 680RD Detection Reagent does NOT work with chicken.



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