Page 97 - GPD-2-2

Gene & Protein in Disease Immunoblotting large and small proteins

Table 1. Recipes for gel preparation The following day, the primary antibody mixture is
removed from the membrane. The membrane is washed at
Final acrylamide Resolving mix Stacking least three times using PBST at room temperature for 5 min
concentration mix
6% 8% 10% 12% 15% each. Then, the membrane is incubated with a secondary
40% AC/Bis mix (mL) 3.0 3.9 5.1 6.0 7.5 0.8 antibody for 1 h at room temperature. Secondary antibody
1.5 M TG 5.2 5.2 5.2 5.2 5.2 - conjugation depends on the developing method. IRDye
(pH = 8.8, mL) antibodies (Li-COR) are visualized using an Odyssey
1 M TG - - - - - 0.8 Imaging System (Li-COR), whereas heavy-chain domain
(pH = 6.8, mL) (HRP)-conjugated secondary antibodies are visualized by
10% SDS (mL) 0.2 0.2 0.2 0.2 0.2 0.06 means of chemiluminescence (WBLUC0100, WBLUF0100,
10% APS (mL) 0.2 0.2 0.2 0.2 0.2 0.06 WBLUR0100, all from Merck Millipore), which is detected
by light-sensitive films (Hyperfilm ECL, 28-9068-37, GE
TEMED (mL) 0.016 0.012 0.008 0.008 0.008 0.006 HEALTHCARE) and further developed using a chemical
Water (mL) 11.4 10.5 9.3 8.4 6.9 4.3 system (Ilford).
V (mL) 20 20 20 20 20 6
F
Recipes for two gels of 1.5 mm width or three gels of 1.0 mm width. 40% 3. Significant modifications for small
AC/Bis mix: Acrylamide/Bis-acrylamide, 40% solution (Sigma, A7168). TG: protein detection
Tris-glycine (Sigma, 108602). Two different TG buffers are prepared. SDS:
Sodium dodecyl-sulfate (Sigma, L3771). SVIP is an 8 kDa lipid-anchored protein composed of
Abbreviations: APS: Ammonium persulfate (Sigma A3678); TEMED: 78 amino acids, which interacts with the endoplasmatic
N,N,N’,N’-Tetramethylenediamine (Bioexpress, M146-50ML). reticulum by adding the N-myristoyl group to glycine-2 .
[4]
The detection of this protein is challenging since it is
to migrate depending on their size. Electrophoresis is not detected following the SWP even after the ectopic
performed in two steps with different constant voltages: overexpression of the protein in a cancer cell line (data not
When protein extracts travel through the stacking phase, shown). Samples containing 40 µg are prepared following
80 V is recommended; once the sample has entered the the SWP. For optimal separation, protein extracts are
resolving phase, the voltage can be increased up to 120 V. loaded into a 15% acrylamide SDS-PAGE gel with 1.5 mm
Typically, electrophoresis is stopped when the dye front of width. These gels are prepared on empty cassettes
reaches the bottom of the gel, which takes 90 to 120 min. (Thermo Scientific, NC2015).
After electrophoresis, the separated protein mixtures Electrophoresis starts at 80 V. Once the front has entered
are transferred to 0.2 µm nitrocellulose membranes the resolving part of the gel, the voltage is increased to 140 V
(GE10600001, Amersham) using Wet/Tank blotting for 75 min. Then, proteins are transferred into a 0.1 µm
systems (1703930, Biorad). Cassettes containing the gel and pore size nitrocellulose membrane. Wet transference is
the membrane, as well as filter papers and sponges, undergo performed at constant voltage, 100V for 12 min, followed
a constant amperage of 350 mA for 1 h in the transfer buffer. by 60 V for 25 min. These steps allow SVIP detection, as
shown in Figure 1C. These SWP changes were also useful for
Under these conditions, proteins migrate from the
gel to the membrane. The transfer buffer is based on a detecting other small proteins, such as histone H3 (15 kDa).
In addition, this protocol was also useful for determining the
running buffer supplemented with 20% of methanol. This protein levels of larger proteins, such as GLUT1 (54 kDa).
step generates heat that may compromise the experiment.
Therefore, the Wet/Tank must be cooled during the whole 4. Significant modifications for large protein
process using a cooling unit inside the tank, covering it detection
with ice, or performing the transfer step in a cold room.
MGA is a 350 kDa estimated weight nuclear protein
2.3. Blocking, antibody incubation, and development composed of 3,065 amino acids [5,6] . MGA can be
The membrane, which contains the separated proteins, is highly modified since it displays 12 sites that can be
blocked by incubating with 5% non-fat skim milk (NFSM; phosphorylated and 47 amino acids that may covalently
232100, Becton Dickinson) in PBS containing 0.01% interact with SUMO-2 protein. The detection of this
Tween 20 (PBST; 85113, Thermofisher) for at least 1 h at protein is arduous since it cannot be detected following the
room temperature on a shaker. Then, the membranes are SWP even after the ectopic overexpression of the protein in
covered by a dilution of the primary antibody in either 5% a cancer cell line (Figure 1D).
NFSM or 5% BSA (antibody dilution is indicated by the For improved immunoblotting, protein samples
manufacturer) and incubated overnight at 4°C on a shaker. are lysed in NP-40 lysis buffer (1% NP-40, 150 mM

Volume 2 Issue 2 (2023) 3 https://doi.org/10.36922/gpd.0547