The Novex NuPAGE Bis-Tris electrophoresis system, now available through InVitrogen, transforms protein gel electrophoresis from a relatively laborious process into a simple, reliable and convenient one. The 'system' consists of precast polyacrylamide gels, running buffers, sample preparation buffers, markers and a gel tank. Although based on traditional methods for separation of denatured proteins on polyacrylamide gels, this patented technology aims at convenience and improved resolution.
The pre-cast gels are pH 6.4 (as opposed to the more typical pH 8.7 of Tris-Cl polyacrylamide gels). The lower pH offers several advantages over traditional gels, perhaps the most appealing and obvious of which is the increased gel stability. Gels can be stored at room temperature for up to one year with no loss in resolution, a result of reduced polyacrylamide hydrolysis. I have kept gels on the bench for several months and noticed no decrease in the quality of results. The lower pH is also supposed to increase protein stability during electrophoresis. Because the protein separation region is at pH 7, as compared to 9.5 of traditional SDS-PAGE, the proteins are less susceptible to modifications such as deamination, alkylation and oxidation.
Modularity is a key feature of the system. Bis-Tris gels are offered in three different acrylamide percentages, and additionally in several formats, chosen depending on sample size, number, etc. In addition to the three gel compositions, two SDS-containing running buffers further influence resolution. Effectively, six different separation ranges can be generated by combining gels and running buffers. For example, the 10 % gel with MES SDS buffer resolves well from 55 kDa to 3 kDa, whereas the same gel with the MOPS SDS can resolve proteins of 100 kDa through to 14 kDa. For very large proteins, Novex Tris-Acetate gels are recommended. Indeed, the complete range of separation is 300-1.5 kDa.
I have always experienced reliable and reproducible results using the sample preparation buffers. The accompanying manual describes in detail correct sample preparation. A 4-times denaturation buffer is available which contains the more soluble lithium salt of dodecyl sulfate (LDS), although precipitates are still present in the buffer when stored in a refrigerator. The pH of the LDS buffer (8.4) obviates asp-pro proteolysis and promotes efficient reduction of disulfide bonds. A 10-times reducing agent (0.5 M DTT) is also available for sample reduction and this is used in conjunction with an anti-oxidant supplemented to running buffers. Of course, if required, non-reduced samples can be electrophoresed. Recommended denaturation conditions are 70C for 10 minutes. Refreshingly, the compositions of all buffers so far mentioned are supplied, which may help save some money.
The final component of the 'system' is the gel tank - the XCell Mini Cell - which is extremely easy to use. The tank works with all Novex gels for protein electrophoresis, so you will not be limited to just the NuPAGE gels. Putting the tank together prior to running gels is very simple, requiring only light pressure to 'snug' everything tight - I have never experienced any leaks. One or two gels can be run at a time, which means up to 28 samples. Certain gels/buffer combinations can be run in as little as 35 mins. I generally stain with Coomassie and reproducibly get sharp, well resolved bands even from crude cell lysates. The attention to detail in this system is exemplified in the SeeBlue Plus2 ready to use markers - they contain landmark that retain their color even after staining (markers do, however, run differently depending upon gel/buffer combinations). Although I have never had to, the gels apparently blot well and a transfer buffer is available.
Personally, the Novex NuPAGE Bis-Tris Electrophoresis System achieves its aim, to simplify protein gel electrophoresis with consistent results. Most importantly, resolution is excellent and band are clean and sharp. In addition to the excellent results, I save a great deal of time and effort.
Peter Haggie, PhD
Post-Doctoral Fellow
University of California, San Francisco