Protocol for blotting onto PVDF membrane
for N-term Edman sequencing
Proteins separated by 1D SDS PAGE can be electroblotted onto PVDF membranes, and the protein bands analyzed by N-terminal Edman sequencing. The blotting protocol below has been optimized for high quality Edman sequencing.
For detection of the protein bands the PVDF membrane can be stained by Ponceau S, Coomassie Blue, Amido Black, and fluorescence Sypro Ruby staining.
Silver staining cannot be used in combination with Edman sequencing!
Western blotting cannot be used in combination with Edman sequencing!
You need the following materials (PVDF membrane and buffer):
Use high quality chemicals and ultra pure water (18.2 Mohm). Wear gloves and use clean staining trays with lids to avoid keratin contamination of the gels and PVDF. Combine any PVDF membrane and blotting buffer below.
- CAPS buffer: (3-cyclohexylaamino-1-propane sulfonic acid) 10 mM CAPS adjusted with NaOH (2N) to pH 11/10% MeOH
- Borate buffer: 50 mM sodium borate, pH 9/ 20% MeOH
- Tris-Glycine buffer: 25mM Tris, 190 mM Glycine, 0.1 % SDS, pH 8.5/ 20% MeO
- NuPAGE Transfer buffer: Invitrogen
PVDF membrane blotting protocol (procedure)
1. Run your 1D or 2D gel.
2. Electroblot using one of the PVDF membranes and buffers above.
Ponceau S Staining
3. If the membrane has dried out, pre-wet it in methanol for a few seconds
4. Stain the membrane in freshly prepared 1% Ponceau S/ 1.0% acetic acid for 2 min.
5. Destain in distilled water until the bands are visible.
3. Stain PVDF with freshly prepared 0.1% coomassie Blue R250 in 40% methanol/1% acetic acid for 30 seconds.
4. Destain with 50% methanol until bands are visible and background clear.
5. Rinse with water.
The PVDF membrane can now be scanned, and protein bands submitted for N-terminal Edman sequencing.
The PVDF membranes and buffers listed in the blotting protocol above generally works well for most proteins. However, some proteins may show poor electroblotting efficiency, and the choice of PVDF membrane, blotting buffer and blotting conditions should be optimized. During optimization it is an advantage to stain the gel after blotting and to use 2 layers of PVDF. Some large proteins (above 80 kDa) may be difficult to get out of the gel, and it can help to add 0.1% SDS to the buffer since SDS increases the mobility of the proteins. The same effect can be obtained by omitting MeOH from the buffer, because MeOH strips SDS from the protein.
Some small proteins (below 15 kDa) may move too quickly out of the gel and through to the first PVDF membrane. In that case, SDS should not be used and the MeOH concentration increased to 20%. Also the gel can be pre-soaked in blotting buffer for 5-10 mins before blotting. Choice of blotting buffers with a neutral pH (Tris-Glycine buffers), may be useful for very basic proteins with high isoelectric points. Basic proteins may be positively charged and the PVDF membrane should be placed on the other or on both sides of the gel. Glycine-containing buffers will give high glycine yield in the first Edman cycle, and the PVDF membrane should be washed extensively after staining.
We have experienced problems and lack of results with the blotting membranes used in the iBlot Dry Blotting system (Invitrogen).
Also problems with the Trans-Blot Turbo Blotting system (Bio-Rad).
The new membrane types used in these systems are optimized for fast and high protein transfer, but are partly dissolved by the Edman degradation chemistry. If transfer membranes can be dissolved in 100% MeOH they are not compatible with Edman sequencing!