Western Blotting Protocol

 

Buffers:

 

TBS:  25 ml of 1 M Tris-7.5, 30 ml of 5 M NaCl, bring volume up to 1000 ml with ddwater

 

TBS-T:  TBS + 0.5 ml of Tween 20

 

5X SDS-PAGE running buffer:  15.1 g Tris base, 94 g glycine in 900 ml water, then add 50 ml of 10% solution of SDS, and adjust volume to 1000 ml with ddwater

 

Blocking solution:  5 g nonfat dry milk in 100 ml of PBS

 

Transfer Buffer:  5.82 g Tris base + 2.93 g glycine + 0.375 g SDS (or 3.75 ml of a 10% solution of SDS) + 200 ml methanol, then bring volume up to 1 liter with ddwater

 

Prepare cell extracts using an appropriate protocol. 

 

  1. Measure total protein in extract by the Bradford method.  Plan on loading approximately 5 ug of protein per lane on the polyacrylamide gel.

 

  1. Add an equal volume of SDS sample buffer and boil the sample on a 100 C block for 5 min.

 

  1. Apply the sample in about 10 ul volume to an appropriate percentage (typically 4-20% gradient) SDS-PAGE mini gel, along with visible protein marker (5 ul) and appropriate positive and negative controls.  Run gel using 1X SDS running buffer.

 

  1. Run gel at 25 mA (constant) for 30 min to 1 hr. (or until blue dye just runs off of the gel)

 

  1. Transfer onto immobilon P membrane using the SD semi-dry transfer apparatus, per apparatus instructions.

 

  1. After transfer, ensure that all the protein has transferred, as assessed by transfer of the visible protein marker onto the immobilon P membrane.

 

7.     Block in 75 ml of blocking solution overnight at 4 C (alternatively, block at room temp for 1 h on a shaking platform).

 

8.     Wash 3 times with TBS-T, each for 10 min.

 

9.     Make dilution of primary antibody (usually 2 ul in 10 ml of TBS), and add to membrane, shake on a platform for 1 h at room temp.  Make sure that membrane is entirely covered with antibody solution.

 

10.  Discard antibody solution and wash with TBS-T 3 times for 10 min each.

 

11.  Make dilution of secondary antibody (usually 1 ul in 10 ml), and cover membrane with antibody solution and shake on a platform for 1 h at room temp.

 

12.  Discard antibody solution, and wash membrane with TBS-T 4 times for 10 min. each.

 

13.  Detection using the ECL/ECLplus kit: