Standard 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
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).
- Nitrocellulose 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.
Western Blotting
- 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.
Using a biotin-conjugated antibody? View protocol here.
