2D Gel Electrophoresis
Two-dimensional gel electrophoresis involves the separation of proteins
based on itys charge and molecular mass to provide a much greater
separation of complex protein mixtures than either of the individual
procedures. The most common two-dimensional technique uses
isoelectrofocusing (IEF) in a tube gel followed by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS-PAGE) in a perpendicular
direction. This combination of isoelectric point (pI) and size separation is
the most powerful tool for protein separations currently available. After
staining, proteins appear on the final two-dimensional gel as round or
elliptical spots instead of the rectangular bands observed on one-
dimensional gels. Although the total separating power of large-format two-
dimensional gels is estimated to be as high as 5000 spots per gel, in
practice a single two-dimensional separation of a complex mixture such as
a whole-cell or tissue extract may produce 1000 to 2000 well-resolved
spots when a sensitive detection method is used.
The most common IEF procedures are based on the use of soluble
ampholytes, which are relatively small organic molecules with various
isoelectric points and buffering capacities. The pH gradient for IEF gels is
produced when the soluble ampholytes migrate in the gel matrix until they
reach their isoelectric point. Because stable pH gradients outside the pH
3.0 to 8.0 range are difficult to create, alternative protocols using non-
equilibrium conditions are required to resolve proteins with pI values
below 3.0 to 4.0 (for acidic proteins) or above 8.0 (for basic proteins). One
of the more important limitations of soluble ampholytes is the difficulty in
obtaining highly reproducible pH profiles, especially when very narrow pH
ranges are needed.
An increasingly attractive alternative to soluble ampholytes is the use of
immobilized pH gradient (IPG) strip gels. In this system, the buffering side
chains are covalently incorporated into the acrylamide matrix, and any pH
range and curve shape can be generated by pouring a gradient gel using
two solutions that differ in ampholyte composition rather than acrylamide
concentration. As with tube gels, the initial IEF separation is followed by a
second separation using SDS-PAGE in a perpendicular direction. The use
of IPG gels has recently increased, for at least three major reasons: many
of the technical problems associated with their use have been solved or
substantially minimized, reproducible pre-made IPG gels are now
commercially available, and lately strong interest has arisen in using two-
dimensional gels for proteome analysis studies (analyzing and comparing
the complete protein profiles of cell lines, tissue samples, or single-celled
organisms).
NOTE: High-purity water (e.g., Milli-Q water or equivalent) and ultra-pure
chemicals are essential for all solutions.
Protein Extraction
Leaf tissues were frozen and ground into fine powder with liquid nitrogen.
The powder was suspended in a pre-cooled (-20ºC) solution of 10%
trichloroacetic acid (TCA) in acetone with 0.07% DTT. The suspension
was incubated at -20ºC for one hour and centrifuged (15 minutes, 35000 g
at 4ºC). The pellet was dissolved in ice-cold acetone containing 0.07%
DTT, incubated at -20ºC for one hour and then centrifuged (15 minutes,
35000 g, 4ºC). (This washing may be continued for two to three times).
The resultant supernatant was discarded and the pellet was freeze-dried.
Proteins were later solubilized in lysis buffer (9M Urea, 4% (w/v) CHAPS,
0.8% (w/v) Biolyte-Ampholyte pH 3-10, 1% (w/v) DTT). Ten-milligram
samples were suspended in 250 µl of lysis buffer and incubated at 37ºC
for one hour with continuous stirring and then centrifuged at 10000 g at
room temperature. The supernatant serves as the protein extract for
analysis. The protein concentrations were measured by the Bradford
method (another protocol).
First and Second Dimension Gel Electrophoresis
For both analytical and preparative gels, the 18 cm IPG strips (pH 4-7)
were rehydrated overnight with 350 µl of rehydration buffer (8M Urea, 2%
CHAPS, DTT (7 mg per 2.5 ml of rehydration buffer) and 0.5% (v/v) IPG
buffer pH 4-7) containing the required quantity of proteins in a reswelling
tray (APBiotech) at room temperature. For analytical and preparative
gels, 100µg and 1.5mg of protein were loaded, respectively. Isoelectric
focusing (IEF) was conducted at 20ºC with a Pharmacia Multiphore II kit.
The running conditions were as follows: 500V for 1 hour followed by
1000V for 1 hour and finally 3000V for 16 hours. The focused strips were
equilibrated twice for 15 minutes in 10ml equilibration solution. The first
equilibration was performed in a solution containing 6M urea, 30% (w/v)
glycerol, 2% (w/v) SDS, 1% (w/v) DTT and 50mM Tris-HCl buffer. The
second equilibration was performed in a solution modified by the
replacement of DTT by 4% (w/v) iodoacetamide. Separation in the
second dimension was performed by SDS-PAGE in a vertical slab of
acrylamide (12% total monomer, with 2.6% crosslinker) using PROTEAN
II MultiCell (BioRad) kit.
Gel Silver Staining
After the termination of the second dimension run, the gels were
immersed in fixative solution (methanol/distilled water/acetic acid,
40/50/10) for one hour. The gels were sensitized by exposure to
thiosulfate reagent (0.02% Sodium thiosulfate), followed by impregnation
with silver nitrate reagent (0.2% silver nitrate and 0.02% of 37 %
formaldehyde) for 30 minutes and developed in developing solution (3%
sodium carbonate, 0.05% formaldehyde (37%), 0.0005% sodium
thiosulfate). The staining reaction was stopped by using 0.5% glycine
solution (for 5 minutes) and gels were rinsed with water several times prior
to densitometry.
Image and Data Analysis
Silver stained gels were scanned using a GS-800 Densitometer (BioRad)
at a resolution of 600 dots and 12-bit per inch. Image treatment, spot
detection and protein quantification were done using Melanie 3 software
(GeneBio, Geneva, Switzerland). Spot detection was carried out on 12-bit
images using optimized parameters as follows: number of smooths, 1;
Laplacian threshold, 5; partial threshold, 5; saturation, 90; peakness
increase, 100; minimum perimeter, 10. Gel matching was performed by
Melanie 3 software and spot pairs were confirmed visually. The scatter
plots between gels of each data point were displayed to estimate gel
similarity or experimental errors and calibration was performed using
fitting report whenever (Melanie 3 user manual). The molecular masses
of protein on gels were determined by co-electrophoresis of standard
protein markers (APBiotech) and pI of the proteins were determined by
migration of the protein spots on 18cm IPG (pH 4-7, linear) strips.