BIOTECHNOLOGY 47
Optimal separation of proteins
Liz Horton and Foner Curtis look at protein separation differences on SDS PAGE dependent on the buffer system
W
hen 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.
Fig. 1. A comparison of several gel buffer chemistries on 10 per cent gels.
Each gel was run using the running buffer and voltage that is noted under the image according to manufacturer’s recommended protocol. Te run time is also noted,
Fig. 2. A comparison of several gel buffer chemistries on gradient gels.
which is the time it took for the ion front to reach the bottom of the gel.
Te marker used is Lonza’s ProSieve Unstained Marker II. Te 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. Te 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. Te run time is also noted, which is the time it took for the ion front to reach the bottom of the gel.
Te marker used is Lonza’s ProSieve Unstained Marker II.
Te gels were stained post-run using ProSieve EX Safe Stain. Te 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
www.scientistlive.com
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