Semi-Dry Transfer to Nitrocellulose

Semi-Dry Transfer to Nitrocellulose

Western Analysis For Alpha-Actin

Semi-Dry Transfer to Nitrocellulose

Proteins are separated by electrophoresis, SDS-PAGE, and individual protein bands can often be identified by using a primary antibody. A primary antibody is a specific immunoglobulin that recognizes and binds to our protein of interest. This process is called Western blotting. For proteins on an electrophoresis gel to be accessible for antibody recognition and binding, they must first be transferred to a nitrocellulose membrane. This process uses a semi-dry method that rapidly transfers four gels at a time.

Materials (For one semi-dry rapid transfer)

  • Pierce G2 Fast Blotter
  • 1 - 6x8.5 cm nitrocellulose sheet
  • 4 - 7x8 cm blotting paper
  • Transfer buffer
  • Blot Roller (or test tube)

Procedure
Multiple gels can be transferred on the same apparatus. Important: Always wear gloves when handling nitrocellulose.

  1. Collect 4 sheets of absorbent 7x8 cm blotting paper (Whatman 3MM or equivalent) and one 6x8.5 cm sheet of nitrocellulose. Cut off a small piece of the upper left-hand corner of the nitrocellulose. This allows you to know the orientation of your blot.
  1. Soak the filter paper and the nitrocellulose membrane in Transfer Buffer for 5 minutes. Use sufficient buffer to completely cover the filter paper and membrane. Note: The membrane should be placed on top while soaking to allow the nitrocellulose to wet to from the bottom-up by capillary action.
  1. While the blotting paper and nitrocellulose membrane are soaking, open the SDS-PAGE cassette and cut off the stacking gel with a metal ruler (see blow).

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  1. Orient the SDS-PAGE gel by making the same small a cut in the upper left-hand corner as on the nitrocellulose membrane. Note: Record the location of all your samples
  1. Carefully rinse the gel with H2O.
  1. In the following order, assemble the module starting from the bottom electrode plate place:
  • Two thick absorbent blotting papers, thoroughly soaked in Transfer Buffer
  • One pre-soaked nitrocellulose membrane
  • One SDS-PAGE gel in the correct orientation
  • Two thick absorbent blotting paper, soaked in Transfer Buffer

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  1. As you add each piece to the stack, carefully remove air bubbles by rolling a glass rod over each layer stack.
  1. After the last module has been assembled gently press the top, the cathode, to lock into place.
  1. Slide cassette into the control unit.
  1. Turn on and select Pre-Programmed Methods in the Main Menu, select the number of gels you wish to transfer, and choose the appropriate program to run (select High MW), and then press start.
  1. After transfer, shut down the unit, turn it off and disconnect the power source.
  1. Carefully disassemble the stack, place the membrane on dry Kimwipes and allow to it dry. The SDS-PAGE gel can now be stained to verify transfer. Pre-stained molecular weight standards ran on the gels should transfer onto the nitrocellulose, and will serve as internal markers for transfer as well as for molecular weight estimation.
  1. Thoroughly rinse both sides of the module with deionized water after every use, as a buildup of salts will damage the unit.

Western Blot Analysis

Materials

  • Blocking solution (5% milk in TBS)
  • Wash Solution (TBST)
  • Primary antibody (1:1,000 anti-alpha actin antibody in 5% milk in TBS)
  • Secondary antibody (1:10,000 anti rabbit-HRP in 5 % milk in TBS)
  • HRP color reagent detection solution.

Procedure

  1. Submerge the membrane in blocking solution.
  1. Incubate at room temperature and shake vigorously overnight. Note: Why are we using milk to block the membrane?
  1. Discard blocking solution and rinse briefly (10 sec) in approximately 20-25 ml of TBST.
  1. Then incubate the membrane in 10 ml primary antibody solution and shake overnight at room temperature.
  1. Briefly rinse membrane in approximately 20-25 ml of Wash Solution. Then add same amount of Wash Solution and shake slowly for 2-3 min.
  1. Discard wash and incubate membrane in 10 ml of secondary antibody solution for 15-20 mins.
  1. Briefly rinse membrane in approximately 20-25 ml of Wash Solution then add same amount of Wash Solution and shake slowly for 2-3 min.
  1. Discard the wash and dry membrane completely before adding the colorimetric solution.
  1. Add 4 ml of HRP color detection solution to the membrane; it could take up to 5 min for the color to develop. Once color develops, go to step 10. Important: Do not over-stain since this will increase the background
  1. Rinse the membrane three times with water and place on dry Kimwipes. Once membrane is dry you can cover it with plastic wrap. Store blot in the dark.
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