A sample Western blot showing Precision Plus Protein Dual Color Marker form Bio-Rad resolved using 10% in-house SDS-PAGE and Lonza ProSieve EX running buffer.
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This product from Lonza is a modified Laemmli SDS-PAGE running buffer that decreases the running time of protein gels to around 30 minutes from the normal 1.5 hrs, without compromising the resolution of separated protein bands.
Protein separation using SDS-PAGE
Rat primary hepatocyte lysate, 1X ProSeive Ex running buffer, 10% in-house gels. (Or any tris-glycine-based pre-cast gel. I have used this buffer with Bio-Rad’s and Life Technology’s Tris-Glycine-based precast gels with similar results).
Dilute the 10X ProSieve EX running buffer to 1X using Millipore-water before use. Prepare SDS-gel or use pre-cast gels. Load samples onto wells. For 10% minigels in the Bio-Rad Mini-PROTEAn TERA system, the running time is around 35 minutes with the voltage set at 200 V.
The buffer level in the outer tank should be up to the sample wells to help prevent the gels from getting hot during the run.
Proteins between 20-250kDa were resolved well. We routinely analyze iNOS, Akt, Gsk3, c-Jun, JNK, b-actin and GAPDH on Westerns from proteins resolved on 10% gel using the Lonza ProSieve EX running buffer.
The 1x buffer can be reused. In our hands, the buffer worked well when used up to 6 times, with no loss of protein resolution. Used 1x buffers were stored at room temperature.