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Western Detection with the Chemi-IR Detection Kit

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Figure 1. Serial dilutions of A431 lysates detected with the Chemi-IR Detection Kit. Serial dilutions of A431 lysates were run by SDS PAGE and transferred to nitrocellulose. The blot was probed with monoclonal anti-p53 followed by Donkey anti-mouse HRP. The blot was detected with IRDye 800CW Rabbit Anti-HRP.

A431_chart

Figure 2. Linearity of the Chemi-IR Detection Kit. Each band of the Chemi-IR Western blot was quantified using Odyssey Imager analysis software. The graph shows the Odyssey detection method is linear to at least 78ng of A431 lysate.

A Western blot that has been incubated with an HRP-conjugated antibody can be detected with IRDye® 800CW anti-HRP. Optimal results are obtained when using a Western blot that has not been processed with a chemiluminescent substrate.

      Notes

    • Ink from most pens and markers will fluoresce on Odyssey®. The ink may wash off and re-deposit elsewhere on the membrane, creating blotches and streaks. Mark with pencil or the Odyssey Pen to avoid this problem. Use pencil for PVDF membrane (wetting in methanol will cause ink to run).
    • Odyssey Blocking Buffer contains 0.1% sodium azide; do not use as HRP-conjugated antibody diluents.
  1. Dilute IRDye 800CW Rabbit anti-HRP 1:1000 (final concentration of 1 µg/mL) in Chemi-IR Diluent. If using large Western Incubation Box included with the kit, dilute 10 µL IRDye 800CW Rabbit anti-HRP into 10 mL of Chemi-IR Diluent. Reminder: Add Tween-20 to the Chemi-IR Diluent before use.

    Important: If using PVDF membrane, add SDS to a final concentration of 0.002 to 0.01% to the Chemi-IR Diluent.

  2. Add diluted IRDye 800CW Rabbit anti-HRP conjugate to blot and incubate for 1 hour at room temperature on platform shaker, protected from light.

  3. Decant diluted conjugate and rinse blot briefly in 15 mL of 1X PBST. Decant. Add 15 mL of 1X PBST and incubate at room temperature on platform shaker protected from light for 5 minutes. Decant. Repeat wash step 2 more times.

  4. 4. Rinse with 15 mL of 1X PBS.

  5. Scan wet on the Odyssey Infrared Imaging System using the Membrane preset with the 800 nm Channel. Start with Scan Intensity set at 6 and adjust as needed.


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