Western Blot Protocol
20X Running Buffer
|Tricine (free base)||71.7 g|
|Tris (free base)||72.6 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|
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|
|Distilled water||350 ml|
Make fresh for each use.
5% non-fat dry milk in TBST
|Carnation non-fat dry milk||50 g|
TBST (Tris Buffered Saline with Tween 20, pH8.0)
|NaC l||8.68 g|
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).
- Cut open the package that contains the gel cassette and drain away the buffer.
- Rinse the wells with distilled water.
- Rinse the wells with fresh 1x running buffer.
- 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.
- Fill the inner core with fresh 1X running buffer. If there are no leaks, fill the outer core with running buffer. Load samples.
- Run the gels at 150V until the dye front reaches the bottom of the gel (approximately 60 minutes).
- Soak nitrocellulose membrane and blotting paper in 1X transfer buffer for at least 5 minutes prior to opening gel cassette.
- Open gel cassettes and place the gel on the nitrocellulose membrane sandwiched between two pieces of blotting paper.
- Place in transfer apparatus and fill with fresh 1X transfer buffer.
- Run transfer apparatus for 60-75 minutes on 35V.
- 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.
- 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.
- Incubate the membrane in diluted primary antibody for two hours to overnight with gentle shaking at room temperature.
- Wash the membrane three times, 10 minutes each time in TBST.
- 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.
- Incubate the membrane in diluted anti-Rabbit IgG-HRP Conjugate or anti-Goat IgG-HRP Conjugate for 60 minutes.
- Wash as directed in step 4.
- Develop blots with substrate solution and place in plastic membrane protector.
- Expose membrane to film or CCD camera.