Note: The following protocols for Western blotting provide general information and conditions that may be applied to most transfer procedures. However, careful optimization should be considered if quantitative recovery of a particular protein is required since blotting efficiency is dependent on buffer composition, blotting time and current, membrane used, etc..

Transfer with a tank blotting system

Transfer buffer*

Ingredient	Amount	Final Stock Concn
Tris-base	3.0 g	25 mM
Glycine	14.4 g	192 mM
Methanol**	100 - 200 ml	10 - 20 %
distilled water	to 1 liter

*cool to 4C before use
**10% methnaol is recommended when using PVDF blotting membranes while 20% is recommended when using nitrocellulose-blotting membranes.

Transfer protocol

Pre-soak the porous pads and two pieces of filter paper cut to the size of the gel (ex. Whatman 3MM paper) in transfer buffer for 5 min
Cut a piece of blotting membrane to the size of the gel and equilibrate in transfer buffer. If a PVDF membrane is used, rinse in methanol for 2 min before equilibration in the transfer buffer for 5 min
Remove the gel from the cassette and equilibrate in transfer buffer for 5 min
Carefully assemble the transfer stack. Remove all air bubbles between the layers. If you do NOT do this then the air bubbles will affect the efficiency of the transfer process and may leave blank spots on the blotting membrane
Place the stack into the tank unit making sure it is in the PROPER ORIENTATION. Fill the chamber with transfer buffer
Transfer at room temperature at 50 mA for 1 to 2 hours.
When the transfer is complete, turn off the power and remove the layers until you reach the blotting membrane
Remove the blotting membrane with a pair of forceps, rinse in distilled water and air-dry for 1 min.

Transfer with a semi-dry system

Transfer buffer

Ingredient	Amount	Final Stock Concn
Tris-base	0.3 g	25 mM
Glycine	1.44 g	192 mM
Methanol	10.0 ml	10% (v/v)
distilled water	to 100 ml

*cool to 4C before use

Transfer protocol
(Note: Wear gloves throughout the procedure)

Pre-soak four pieces of blotting membrane to the size of the gel and equilibrate in transfer buffer. If a PVDF membrane is used, rinse it in methanol for 2 min before equilibration in the transfer buffer for 5 min
Remove the gel from the cassette. Carefully assemble the transfer stack on the anode (+). Remove all air bubbles between the layers. Air bubbles will affect the efficiency of the transfer process and may leave blank spots on the blotting membrane
Transfer at room temperature at 50 mA for 1 to 2 hours.
When the transfer is complete turn off the power and remove layers until you reach the blotting membrane
Remove the blotting membrane with a pair of forceps, rinse in distilled water and air-dry for 1 min

Monitoring of protein transfer

The efficiency of transfer can be monitored using prestained protein markers. Alternatively, the extent of protein transfer can be determined by staining the polyacrylamide gel after the trasnfer is complete or by staining the proteins directly on the blotting membrane. Proteins on solid support can be stained with dyes such as India ink, Amido Black or Coomassie Blue. A recommended stain is Ponceau S, which is reversible. The detection limit is 1 to 2 micrograms.

Ponceau S staining for proteins on solid support

Ingredient	Amount	Final Stock Concn
Ponceau S	0.2 g	0.2% (w/v)
Glacial Acetic Acid	1 ml	1% (v/v)
distilled water	to 100 ml

Protocol

If the blotting membrane is dry, rehydrate it with water (or methanol, if PVDF) for 5 min
Stain the blotting membrane under constant shaking with the Ponceau S solution for 5 min
Destain the membrane under constant shaking with distilled water
If required, wash the blot with 0.1N NaOH to remove the stain completely