How do I optimize electroblotting conditions onto PVDF membrane for N-terminal Edman sequencing? Our 12 kDa protein separates well on 1D SDS PAGE but does not appear on the PVDF blot for N-terminal sequencing. We are using the NuPAGE Transfer buffer with 20% MeOH from Invitrogen. What can we do to optimize the blotting conditions?
The blotting buffer generally works well for most proteins. However, some proteins may show poor electroblotting efficiency. Thus you should optimize your choice of PVDF membrane, blotting buffer and blotting conditions. 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 is 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 try pre-soaking the gel 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. Therefore, you should place the PVDF membrane on the other or on both sides of the gel. Glycine-containing buffers give a high glycine yield in the first Edman cycle. To avoid this, you should wash the PVDF membrane extensively after staining.