1. Lab#8: pGLO SDS PAGE Extension
Dr. Ricardo Chiesa
The main objective of this laboratory was to separate all proteins expressed in
E.coli after transformation, a step done in the previous lab. We labeled four screw capped
microtubes respectively as follows; white no heat, green no heat, white+ heat, green+
heat. We added Laemmli buffer to the two no heat samples. With an inoculation loop, we
scrape sufficient mass of pGLO containing bacteria colonies from both positive pGLO
dishes. We followed the protocol step by step to create our samples. After the samples
were ready, with a micropipette we prepared the polyacrylimide gel for running. This was
a double sample gel since we used the same samples to conduct two tests. We divided the
gel in two parts, from lane 1-5 the gel was stained with coomassie blue and from lane
6-10 unstained. As a result, lane 8 from the unstained SDS PAGE gel fluoresced at 37kD
whereas in lanes 9 and 10 proteins are completely denatured and no signal is present. On
the other hand, the coomassie stained gel demonstrated a variety of E. coli proteins
extending in size from 10-160 kD. GFP is completely denatured at 27kD and is partially
denatured at 37kD. This is shown in lane 3, and lane 5 with just the completely denatured
band at 37kD. Now we know the size of GFP that is 37 kD and how it is used as a
marker.