Protocol for semidry transfer after Gel Shift assay using Chemiluminescence

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suma
suma's picture
Protocol for semidry transfer after Gel Shift assay using Chemiluminescence

Hey,
can somebody send me the protocol for semi dry transfer after Gel shift assay using Chemiluminescent method.

What transfer buffer I should be using?

Thanks

Sr

Edward Dougherty
Edward Dougherty's picture
1. Cut 4 pieces of Whatman's

1. Cut 4 pieces of Whatman's paper
2. After running your gel, separate the gel plates leaving the gel stuck to the rear gel plate and place one piece of Whatman's paper carefully over the gel so that the paper lies flat against the gel with no creases or air pockets (To do this easily, lightly fold the paper into a "U" shape with your hands. Then, push the bottom curve of the "U" against the middle of the gel and carefully let down the sides of the "U" against the gel). Then, place a second piece of Whatman's paper beneath the first.
3. Carefully remove the gel from the other gel plate, leaving the gel stuck to the Whatman's paper.
4. Place the gel+Whatman's papers into a tray containing 0.5x TE buffer.
5. Place the nitrocellulose membrane over the gel (pre-soak the membrane as directed by the manufacturer), then two more pieces of pre-soaked Whatman's paper.
6. Push down carefully to ensure no air bubbles are trapped in your gel sandwich.
7. Transfer the gel sandwich to your semidry apparatus
8. Transfer for 30-60 min at 300 mA (Current and time may differ depending on your experimental needs)