dodecyl sulfate polyacrylamides (SDS-PAGE) gels are most commonly used for separating
proteins on the basis of size (Laemmli 1970). In these gels, disulfide reducing agents such
asb-mercaptoethanol or DTT are added to denature the proteins intopolypeptides, and SDS
swamps the polypeptides with a net negative charge so that they migrate toward the anode
(Laemml 1970; Memelink et al. 1994). The proteins are transferred to polyvinylidene
fluoride (PVDF) membrane by electroblotting, the membrane is incubated with an antibody
raised to the transgenic protein, and the antibody binds primarily through hydrogen bonds
(Memelink et al. 1994; Sambrook and Russell 2001). The antibody is detected with an
enzyme conjugate secondary antibody that causes a color or chemiluminescent change
detectable on film (Memelink et al. 1994) (Fig. 11.11). Protein bands can be semiquantified
by band intensity or relative to a standard protein.
11.5 Digital Imaging
In the olden days of molecular biology (pre-1990s), simple nondigital photographs were
taken to document raw data from gels and blots. No one does that anymore with the
advent of digital imaging. Most journals have guidelines stating which software imaging
adjustments are acceptable for enhancing digital images of nucleic acid and protein blots
to avoid appearances of research misconduct. For example, Rossner and Yamada 2004
offer a set of guidelines for the Journal of Cell Biology(http://www.jcb.org/cgi/
content/full/ 166 / 1 /11). Linear adjustments to brightness and contrast must be made to
the entire gel image—never to only a portion of the gel, and never to obscure or alter
the original data. For example, any nonlinear gamma adjustments must be disclosed in
the figure legend. Selective removal of background within lanes must be performed judi-
ciously, if at all. It is prudent to consult the specific journal for its instructions in handling
digital images.
11.6 Phenotypic Analysis
After screening and molecular characterization, transgenic plants should be grown and their
phenotypesassessed to determine whether they differ from wild-type. The phenotype is the
genetic make-up (genotype) as influenced by the environment. Traits altered with respect to
the SM used in transformation experiments such as resistance to herbicides and resistance to
antibiotics should be tabulated. A transgenic plant expressing a new GOI may also have an
altered phenotype with respect to seedling emergence, growth habit, days to flower, days to
maturity, seed color, disease resistance, and other parameters, in comparison to wild-type.
11.7. CONCLUSIONS
Initial screens on selective media, with ELISAs or PCR will determine whether one has
putative transformants. Copy number Southern gel blots will show transgene integration.
Real-time PCR can augment but not substitute for copy number Southern gel blots.
Analysis of intact genes will show whether the GOI or SM has been transferred in its
entirety to the plant or is truncated. Northern gel blots, RT-PCR, and western gel blots
11.7. CONCLUSIONS 287