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Supplemental Product Information:

Product Information

Components : 10X Negative Staining Solution A, 10X Negative Staining Solution B, 10X Negative De-Staining Solution C
Size : 20 tests

Description

Imidazole-Zinc Reverse Staining is a detection method for SDS-PAGE separated proteins, in which only regions of gel not containing proteins are stained white, while protein-containing regions remain transparent. Bands are visualized by placing the gel against a dark background. Destaining can be achieved using the Negative De-Staining Solution C, allowing proteins to be eluted or transferred to a membrane.

Storage

Store at room temperature and used within 12 months.

Direction for Use

Preparation (For 90 x 90 x 1mm gel)
The amount of reagent to adjust depends on the gel size.

Solution A : Prepare Solution A by mixing 5 mL of 10X Negative Staining Solution A with 45 mL of de-ionized water. (Note: Precipitates formed during storage should be redissolved at 25 - 30°C prior to use.)
Solution B : Prepare Solution B by mixing 5 mL of 10X Negative Staining Solution B with 45 mL of de-ionized water. (Note: This solution contains zinc sulfate. Check your local laws for disposal of Zinc.)
Solution C : Prepare Solution C by mixing 5 mL of 10X Negative De-Staining Solution C with 45 mL of de-ionized water.
Preservative solution : Prepare by mixing 200 μL of 10X Negative Staining Solution A with 50 mL of de-ionized water.

1. Place gel in a properly-sized container with 50mL of deionized water and shake for 5 - 10 minutes.
2. Remove deionized water, add 50mL Solution A, and shake 5min.
3. Remove Solution A and rinse the gel 3 times (10 seconds) with 50mL of deionized water. (Note: Rinsing for too long will lead to poor staining results.)
4. Place gel in another container, add 50mL Solution B, and shake for 30 - 100 seconds. Note: This step must not be extended longer than 100s or band overstaining and loss of the image will occur.
5. Transfer the gel to the container used in step 3, add 50mL deionized water, and rinse as soon as the bands appear. Rinse the gel 3 times with water for 30 – 60 seconds. Photograph immediately afterwards.
6. Photograph the gel. It is recommended that the gel be photographed on a black tray. Note: Gels can be stored in Preservative solution, but should be photographed as soon as possible. Stained gels cannot be dried and stored.
7. Remove deionized water, add 50mL Solution C, and shake until stain is removed.
8. Remove Solution C and rinse the gel 3 times (30 seconds) with 50mL of deionized water.
9. After destaining, proteins are free of dyes and other contaminants. As such, they can be transferred to a membrane for Western blotting or eluted from excised gel pieces.