Gel Stain Reagents (Other)

This product is a kit for negative staining of proteins in the gel, after SDS-PAGE. When the gel is stained with this product, the gel becomes cloudy, but no proteins are stained. Since no proteins are modified by the dye, the product is suitable for MS analysis and peptide mass fingerprinting. The lower limit of detection of proteins is approximately 5 ng.

Kit composition

• Staining solution A・・・500 mL
• Staining solution B・・・500 mL
• Destaining solution・・・500 mL

Staining method

1. After SDS-PAGE, soak the gel in a clear container containing 25 mL of Staining solution A and shake for 5 to 10 minutes.
2. Wash the gel with 50 mL of distilled water for 5 to 10 seconds; repeat three times.
3. Add 25 mL of Staining solution B and shake for approximately 10 to 60 seconds while confirming the staining status on black paper.
4. Wash the gel with distilled water for 1 minute; repeat three times.

Destaining method

1. Add 25 mL of destaining solution and shake for approximately 5 minutes until the gel becomes transparent.
2. Wash the gel with 50 mL of distilled water for 1 minute; repeat three times.
   * Destaining can be followed by Western blotting. In this case, follow the Western blot procedure.

This product is used in reversible total protein staining for Western blotting and serum protein staining in cellulose acetate membrane electrophoresis.

Usage (Western Blotting)

1. Staining: After protein transfer, soak the membrane in "0.1 w/v% Ponceau 3R, 5 v/v% acetic acid" solution or Ponceau-3R Stain Solution and shake until bands become visible (approximately 2 to 10 minutes).
2. Destaining 1: Shake the membrane with water, PBS-T, or TBS-T until the background intensity is reduced (approximately 5 minutes). Take images here if necessary. This step is unnecessary if the background does not matter.
3. Destaining 2: Shake the membrane in 0.1 mol/L NaOH until it is completely destained (approximately 30 to 60 seconds).
4. Washing: Shake the membrane in water for approximately 2 minutes.
5. Block the membrane and proceed to the antibody reaction.

This product is used to detect proteins transferred to the membrane or attached to equipment.