Chapter 1, The Reptilian Spectacle - UWSpace - University of ...

for 15 minutes to remove all residual Tween 20. Following this, either a chemiluminescent reagent
(Amersham ECL Prime) was applied for 3 minutes and the resultant luminescence either photographed
or scanned using a chemifluorescence imager (Storm 840, Molecular Dynamics), or the membrane was
incubated with a colorimetric reagent (Opti-4CN, Bio-Rad) for 1-10 minutes, rinsed for 15 minutes in
deionized water, and documented with a desktop scanner.
4.2.5 2-Dimensional Electrophoresis
2-Dimensional electrophoresis was performed on coachwhip spectacle and parietal scales to expose
isoforms with varying pI’s.
4.2.6 Isoelectric Focusing (IEF)
Samples were diluted with rehydration buffer (7 M urea, 2 M thiourea, 5% glycerol, 20mM DTT, 0.5%
immobilized pH gradient (IPG) buffer ampholytes (Bio-Lyte 3/10, Bio-Rad) 0.005% bromophenol
blue) and applied to 7 cm nonlinear pH 3-10 IPG strips (Immobiline DryStrip, GE Healthcare) which
were then left to rehydrate for 12 hours. IEF was performed using an Ettan IPGPhor II (GE
Healthcare) according to the following protocol: 500 V for 30 min, gradient to 1000 V for 30 min,
gradient to 5000 V for 90 min, 5000 V for 35 min, 500 V up to 12 hours until the strips were removed.
On completion, the strips were stored at -20ºC until the second dimension could be run. Due to the
high density of ß keratins aggregating at similar pH, two separations with different amounts of loaded
protein (3.75 µg and 12.5 µg) were performed for each sample to adequately resolve all proteins.
4.2.7 2nd Dimension (SDS-PAGE)
The frozen IPG strips were thawed and immersed for 15 minutes in equilibration buffer (6 M urea, 50
mM Tris-HCl, 30% glycerol, 2% SDS, 0.005% bromophenol blue, 2% w/v DTT), followed by another
15 minutes in the same buffer with 4% w/v iodoacetamide replacing the DTT. The strips were then
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