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HomeGel ElectrophoresisReadyBlue™ Protein Gel Staining Protocols

ReadyBlue™ Protein Gel Staining Protocols

ReadyBlue™ Protein Gel Stain is a rapid and sensitive colloidal Coomassie® stain for polyacrylamide gels. The stain is provided as a ready-to-use solution, allowing for a fast and simplified protocol. No prior fixing step is necessary. For convenience, ReadyBlue™ gel stain can be stored at room temperature. For additional protein gel staining products and protein staining gel needs, please visit our Protein Electrophoresis Gel Stains resource page.

Standard ReadyBlue™ Protein Gel Staining Protocol

  1. Pour stain into a clean, detergent-free container. Use approximately 25 mL for mini gels (e.g. 8 X 10 cm gels). For larger gels use a volume of staining solution equal to approximately five times the gel volume. Using too little staining solution can reduce sensitivity.
  2. Place gel in staining solution and incubate with gentle agitation on a platform shaker for a minimum of one hour.
  3. For higher intensity, stain for 1-2 hours. Gels will not over-stain if left in staining solution overnight.
  4. Remove the stain and wash gels in deionized water to remove any background staining and for gel storage.
12% mPAGE® Bis-Tris gel loaded with 2-fold serial dilutions of BSA and run with MOPS buffer at 200V for 30 minutes

Figure 1.12% mPAGE® Bis-Tris gel loaded with 2-fold serial dilutions of BSA and run with MOPS buffer at 200V for 30 minutes. Gel was imaged with white light and no destaining at 5, 15, 30, and 60 minutes. Gel was also imaged after destaining for 60 minutes with white light and with a LI-COR Odyssey® in the 800 channel for fluorescence.

Microwave Gel Staining Protocol (Optional)

Adding a brief microwave step can decrease staining time and allow for a quick evaluation of staining efficacy. For maximum sensitivity, use the standard protocol.

  1. Immerse the gel in ReadyBlue™ Protein Gel Stain, in a clean, detergent-free, microwave-safe container and place in microwave for a maximum 60 seconds at full power.
  2. Remove the container from the microwave. Wash gels for 120 seconds in deionized water at room temperature to remove any background staining. Do not microwave gel to remove background.

NOTE: A minimum one-hour full stain is recommended before storing the gel in water.

Protocol for Destaining Bands Prior to Mass Spectrometry Analysis

  1. Use standard gel staining protocol.
  2. Excise the protein band of interest and place in a clean Microfuge® tube.
  3. Add 1 mL of 30% ethanol or acetone.
  4. Incubate for 20 minutes (Increasing temperature to 60 °C–70 °C decreases time needed for destaining).
  5. Decant supernatant and repeat step 3 and 4 a minimum of 3 times or until the gel fragment is clear
  6. Continue with required mass spectrometer sample preparation steps.

For additional information and resources in support of your mass spectrometry workflow needs, please visit our Protein Mass Spectrometry resource hub.

ReadyBlue™ Protein Gel Staining Storage and Stability

ReadyBlue™ Protein Gel Stain features a shelf-life of up to 18 months from the date of manufacture when stored at 2–8 °C or room temperature and protected from light. Stain may be reused up to three times, however there may be a reduction in sensitivity with each subsequent use because of loss of dye. If the stain is reused, then it is the responsibility of the user to confirm proper storage conditions and verify performance.

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