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Optimal separation of proteins

20th May 2013


Liz Horton and Foner Curtis look at protein separation differences on SDS PAGE dependent on the buffer system.

When choosing an SDS PAGE gel to run, in order to get optimal separation of the proteins of interest it is important to consider both the gel percentage as well as the buffer system.

Here, we present separation data using protein markers on the new PAGEr EX Gels run using ProSieve EX Running Buffer compared to similar standard gels with well-known or alternate buffer systems, along with Lonza's PAGEr Gold run with traditional Tris-glycine SDS.

In Fig. 1, we have a comparison of several gel buffer chemistries on 10 per cent gels and separation range similarities and/or differences of protein sizes relative to the PAGEr EX Low/Mid Gels.

Each gel was run using the running buffer and voltage that is noted under the image according to manufacturer's recommended protocol. The run time is also noted, which is the time it took for the ion front to reach the bottom of the gel.

The marker used is Lonza's ProSieve Unstained Marker II. The gels were stained post-run using ProSieve EX Safe Stain. With the Tris-glycine SDS on PAGEr Gold and Company B gels, and the Company L Bis Tris gels run with MOPS, the 10 kDa is lost in the ion front. The PAGEr EX Gel shows the lowest band and a greater separation up to the 50-75kDa range relative to the others.

In Fig. 2, we look at a comparison of several gel buffer chemistries on gradient gels and separation range similarities and/or differences of protein sizes relative to the PAGEr EX Mid/High Gels.

Each gel was run using the running buffer and voltage that is noted under the image according to manufacturer's recommended protocol. The run time is also noted, which is the time it took for the ion front to reach the bottom of the gel. The marker used is Lonza's ProSieve Unstained Marker II.

The gels were stained post-run using ProSieve EX Safe Stain. The PAGEr EX Mid/High Gel range gives optimal separation in the 50-200kDa range yet still has greater separation from 50kDa down to 10kDa, making this a versatile gel for analysis of a broad range of sizes.

Conclusion

PAGEr EX Gels cover a broad range with optimal separation in only two gel formats and an extended one year shelf life.

When combined with ProSieve EX Running Buffer, Transfer Buffer and Stains, the process is reduced from five hours down to one hour in total with no compromise.

For more information at www.scientistlive.com/eurolab

Liz Horton and Foner Curtis are with Lonza Walkersville, Inc, Walkersville, MD, USA. www.lonza.com





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