One of the most important aspects of specific transcript quantitation is use of the appropriate
statistical analyses (Yuan et al. 2006).
11.4.4 Transgene Expression: Translation: Western Blot Analyses
Protein gel blot analyses or western blots show in a semiquantitative manner that the intro-
duction and transcription of a GOI is causing a production of a specific recombinant protein
in the transgenic plant. The researcher can see if the intact protein is produced or whether it
is not processed correctly. In western blotting (Burnette 1981), total protein is isolated and
fractionated on a polyacrylamide gel, transferred to a membrane, and then detected with
primary and secondary antibodies (Reece 2004) (Figs. 11.10 and 11.11). Sodium
Figure 11.10.The process of western blot analysis. (a) polypeptides are separated according to size
on a polyacrylamide-SDS gel; (b) the polypeptides are transferred to a PVDF membrane; (c) the mem-
brane is incubated with an antibody raised to the transgenic protein, and the antibody is detected with
an enzyme conjugate secondary antibody that causes a color or chemiluminescent change detectable
on film.
Figure 11.11.Western blot. This experiment used a GFP-specific polyclonal antibody to detect GFP
in transgenic plants. A semiquantitative assay can be performed by running purified GFP at various
concentrations, in this case, from 2 to 20 ng (see the first three lanes to the right of the protein size
standard). The next lane is from a mGFP5-ER transgenic plant. The next lanes are from individual
transgenic plant events with an earlier GFP variant—mGFP4. Note that some transgenic events are
extremely low-expressing and that there are two bands where only one is expected. (Data courtesy
of Staci Leffel and Neal Stewart.)
286 TRANSGENIC PLANT ANALYSIS