Western Blot Protocol

This protocol outlines the reagents, buffer and step-by-step procedure to be used with western blot experimentation. This outlines the steps for electrophoresis in SDS-polyacrylamide gel and transfer to nitrocellulose.

Materials Needed:

20X Running Buffer

Tricine (free base) 71.7 g
Tris (free base) 72.6 g
SDS 10.0 g
Sodium Bisulfite 2.5 g

Adjust to 500 ml with ultra pure water.

Store at 4 °C. For 1X Running Buffer, add 10 ml of 20X Running Buffer to 190 ml of distilled water.

10X Transfer Buffer

Tris (free base) 15.2 g
Glycine 72.1 g
SDS 5.0 g

Ultra pure water to 500 ml

10X Transfer Buffer is available from PAGE gels (Cat# CB82500)

Store at 4 °C.

1X Transfer Buffer

10X Transfer Buffer 50 ml
Methanol 100 ml
Distilled water 350 ml

Make fresh for each use.

5% non-fat dry milk in TBST

Carnation non-fat dry milk 50 g
TBST 1 liter

TBST (Tris Buffered Saline with Tween 20, pH8.0)

Tris 6.1 g
NaC l 8.68 g
Tween-20 500 mcl

Adjust the volume to 1 liter with distilled water. Adjust pH to 8.0 with HCL.

Store at 4-25 °C.

Electrophoresis in SDS-Polyacrylamide Gel and Transfer to Nitrocellulose:

Many of the reagents for these procedures are commercially available. Sources that are preferred by Bethyl Laboratories, Inc. are:

  • Polyacrylamide gels (4-12% Tricine), running buffer and transfer buffer from PAGEgel (San Diego, CA).
  • SeeBlue2 and HiMark molecular weight markers - Invitrogen (Carlsbad, CA).
  • itrocellulose membranes - Invitrogen (Carlsbad,CA).
  1. Cut open the package that contains the gel cassette and drain away the buffer.
  2. Rinse the wells with distilled water.
  3. Rinse the wells with fresh 1x running buffer.
  4. Place the gels on the buffer core so that the shorter plates face inward. If only using one gel, use a buffer dam to seal the other side.
  5. Fill the inner core with fresh 1X running buffer. If there are no leaks, fill the outer core with running buffer. Load samples.
  6. Run the gels at 150V until the dye front reaches the bottom of the gel (approximately 60 minutes).
  7. Soak nitrocellulose membrane and blotting paper in 1X transfer buffer for at least 5 minutes prior to opening gel cassette.
  8. Open gel cassettes and place the gel on the nitrocellulose membrane sandwiched between two pieces of blotting paper.
  9. Place in transfer apparatus and fill with fresh 1X transfer buffer.
  10. Run transfer apparatus for 60-75 minutes on 35V.

Western Blotting:

  1. Remove the membrane from the transfer apparatus and place in 20 ml of 5% non-fat dry milk in TBST for one hour, with gentle shaking.
  2. Dilute the primary antibody in 15 ml of 5% non-fat dry milk in TBST. For best results, the optimal dilution of antibody should be empirically defined.
  3. Incubate the membrane in diluted primary antibody for two hours to overnight with gentle shaking at room temperature.
  4. Wash the membrane three times, 10 minutes each time in TBST.
  5. Dilute the secondary HRP conjugated antibody (Bethyl anti-Rabbit IgG-HRP Cat. #A120-101P or Bethyl anti-Goat IgG-HRP Cat. #A50-100P) in 15 ml of reconstituted 5% non-fat dry milk in TBST. For best results, the optimal concentration of the secondary HRP conjugated antibody should be empirically defined.
  6. Incubate the membrane in diluted anti-Rabbit IgG-HRP Conjugate or anti-Goat IgG-HRP Conjugate for 60 minutes.
  7. Wash as directed in step 4.
  8. Develop blots with substrate solution and place in plastic membrane protector.
  9. Expose membrane to film or CCD camera.