Blotting
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Following electrophoresis, Western blotting involves the transfer and immobilization of proteins from a gel to a solid support membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). Blotting allows the use of immunodetection, which would otherwise be difficult to perform using gels directly.
The principle of blotting relies on the same electro-mobility properties as those that separated proteins on the polyacrylamide gel during electrophoresis. The SDS-bound proteins are negatively charged and migrate toward the positively charged anode. However, the gel, the membrane, and the electrodes are assembled in sandwich so that proteins will move laterally, halting their course on the membrane where they print their migration pattern.
GE Healthcare provides two instrument categories for blotting:
- Tank blotting instruments for high performance and transfer conditions flexibility.
- Semi-dry blotting instruments requiring low voltages and minimal buffer volumes.
GE Healthcare also offers a great selection of related products for blotting.
Factors to consider when performing Western blotting
Parameters such as sample characteristics, membrane type, gel pore size, and the transfer buffer used all contribute to the transferability of macromolecules, and should be kept in mind when developing a protocol. Very small molecular species, for instance, migrate quickly but often do not bind as well as larger molecules. The rate of elution is also affected by the pore size of the gel and the orientation of the molecules.
- The blotting membrane type and the transfer characteristics
The degree to which molecules bind to the membrane is influenced by membrane characteristics such as pore size and type, and buffer characteristics such as pH, salt type, salt concentration, and the presence of detergents such as SDS.
Conditions required for efficient elution may not coincide with optimal conditions for binding. To find the optimum conditions for transferring your sample, one must balance these effects: if the sample elution rate is slow, a longer transfer period may be required. However, low voltage transfers for longer periods do not offer much improvement. You may also try different buffer conditions if sample binding is inadequate.
- Gel equilibration in transfer buffer
If the transfer buffer system is different from the electrophoresis buffer system, the gel should be equilibrated with the transfer buffer before the transfer to ensure swelling or shrinking occurs before the gel contacts the transfer membrane. If this step is skipped, band distortion or loss of resolution could result.
- Methanol in transfer buffer
Methanol in the Towbin buffer system is necessary to achieve efficient binding to nitrocellulose and PVDF membranes. Methanol improves binding in part by decreasing the SDS affinity to immobilized proteins. However, it may also cause a gel to shrink, so the elution rate decreases.
Membrane type | Suggested Methanol % |
Nitrocellulose | ≤ 20 |
PVDF | ≤ 15 |
|
- Sodium Dodecyl Sulfate (SDS) in transfer buffer
It was reported that a low concentration of SDS (0.02-0.1%) improves the transfer of protein from an SDS-containing gel.
Click here for more information about the recommended buffer systems for GE Healthcare blotting instruments
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