Preparation of SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis)
Samples were analyzed by SDS-PAGE followed by immunoblotting. First, 10% SDS-PAGE gels were custom made with a 10% resolving and 3% stacking gels using the recipe listed in Tables 1 and 2. Bio-Rad MiniProteon 3 gel casting system containing 0.75
mm/ 1 mm spacer and small plate was used for all SDS-PAGE. Typically, 0.75 mm spacer plate and small plates are assembled using clamp assemblies and gasket in a gel- casting stand. The 1 mm spacer plates were used to run gels that required larger sample amounts (up to 30 µL) for analysis. After mixing all the reagents in the order listed in Table 1, the resolving gel was slowly pipetted in the gel cassette followed by adding 1 mL of isopropanol to ensure a flat surface for the resolving gel. The resolving gel was typically polymerized within 20 min upon casting. The isopropanol was washed out with water and residual water was removed using filter paper. The 3% stacking gel was carefully pipetted on top of the resolving gels and immediately a 10- or 15-well comb was set in place for 20 min. Once polymerized the comb was removed and gel was placed in a buffer tank containing 1× SDS Running buffer (25 mM Tris Base, 192 mM Glycine, 0.1% SDS).
Table 1: 10 % SDS-PAGE separating gel recipe.
10% separating gel 1 gel
0.75M Tris HCl pH 8.8 2.5 mL
10% SDS 150 µL
30% Acrylamide-0.8%Bisacrylamide(@ 4˚C) 1.67 mL 10% Ammonium per sulfate (@ 4˚C) 30 µL
Sterile filtered water 0.75 mL
TEMED (Tetramethylethylenediamine) (@ 20˚C) 5 µL
Table 2: 3% SDS-PAGE stacking gel recipe.
3% stacking gel 1 gel
10% SDS 22.5 µL 30% Acrylamide-0.8%Bisacrylamide(@ 4˚C) 200 µL 10% Ammonium per sulfate (@ 4˚C) 25 µL
Sterile filtered water 1000 mL
TEMED (Tetramethylethylenediamine) (@ 20˚C) 2.5 µL
Sample preparation for SDS-PAGE
Samples were prepared differently depending upon the experiment. Typically, for sample preparation following an immunoprecipitation (IP) experiment, proteins were eluted by adding 20 µL of 2× sample buffer (8% SDS, 10% glycerol, 0.7 M β- mercaptoethanol, 37.5 mM Tris-HCl, pH 6.5, 0.003% bromophenol blue) to IP samples. Samples are incubated for 5 min at 100˚C followed by centrifugation for 30 sec at 13,000 rpm using a Spectrafuge 24D microcentrifuge (Labnet International, Inc.).
In signaling or degradation experiments, whole cell lysates were prepared by lysing cells in 300 µL of 2× sample buffer. Samples were either stored at -20˚C or immediately processed for gel electrophoresis. Samples were sonicated using a Branson 450 digital sonifier containing a convertor and microprobe set at 11% amplitude for 10 sec. Samples were then centrifuged for 30 sec at 13,000 rpm using a Spectrafuge 24D microcentrifuge (Labnet International, Inc.). After sample preparation, 10 µL of sample was loaded onto a 10% SDS-PAGE gel. To monitor electrophoretic separation and molecular weight sizing protein standard (Bio-rad laboratories.Inc) was diluted 1 µL in 9 µL of 2× sample buffer and loaded along with the samples. The samples were electrophoresed at 160V for 1 h in the Mini-PROTEAN 3 system (Bio-Rad).
Electrophoretic Transfer of proteins onto nitrocellulose membrane
Gel was removed from the SDS-PAGE set up and placed in 1× Transfer buffer (0.025M Tris-Base, 0.192M Glycine, 20% (vol/vol) methanol and water). Gel-membrane sandwich was made using fiber pads, 0.45 µM nitrocellulose membrane (Bio-rad), gel and whatman filter paper. In the transfer cassette, fiber pads are placed first on the grey side of the cassette followed by placing the whatman filter paper. Gel is then placed on the filter paper followed by placing the nitrocellualose membrane and fiber pads on the gel. Cassette is closed carefully by removing bubbles to ensure efficient transfer of proteins. Cassette is then placed in transfer tank containing 1× transfer buffer, ice-pack and magnetic needle with gel side facing the negative electrode. Transfer tank was placed on the stir plate and transferred for an hour at 100V. After the transfer, the membrane was removed and incubated with ponceau for 5 min to stain proteins. Membranes were quickly rinsed in approximately 50 mL water to remove background binding and to determine the efficiency of protein transfer. Membrane was labeled with ethanol free marker with name and date of experiment.
Immunoblotting
Membrane was rinsed once with approximately 50 mL of 1× TBST (Tris- Buffered Saline and Tween 20) (50 mM Tris HCl pH 7.4, 150 mM NaCl, 0.1% Tween 20) to remove the bound ponceau staining. Membrane was then incubated with 5% milk made in 1× TBST for 30 min on a rocking platform VWR (04033ID). In order to immunoblot with pAkt-T308 antibody, membrane was incubated with western blot
blocking reagent from Roche (Mannheim, Germany). After 30 min of blocking, membranes were then incubated with primary antibodies made in 5% milk made in 1× TBST or blocking reagent based on the experiments for 1 hr or 12-16 hr at 4˚C on the rocking platform. After the incubation, membrane was washed 3× with 1× TBST on the rocker for 5 min each at RT. 5% milk containing species-specific Horse-Radish Peroxidase (HRP) conjugated secondary antibody was added to the membrane for 30 min followed by 5× washes with 1× TBST for 10 min each at RT.
Preparation of chemiluminescent substrates
To analyze the signals on the membrane, we used super signal- west –DURA enhanced chemiluminescent substrate (Thermo Scientific). To detect tagged transfected proteins or loading control proteins such as actin or tubulin, a chemiluminescent substrate prepared in the laboratory was used. The in house chemiluminescent substrate was prepared by mixing solution 1 (2.5mM 3-Aminophthal Hydrazide and 0.45mM p- Coumaric acid in 0.1mM Tris pH 8.8) and solution 2 (0.02% Hydorgen peroxide in 0.1M Tris pH 8.8) in 1:1 ratio. After the final 1× TBST wash, membranes were then removed from the 1× TBST, dabbed to remove excess buffer and incubated with DURA or lab made chemiluminescent substrate for 5 min. In the dark room, protein bands were visualized by exposing the autoradiography films on the membrane for various exposure times followed by developing films in a SRX-101A automated medical film processor (Konica Minolta Medical and Graphic.Inc).