Ettan dalt six device

Six Immobiline Dry Strips (24 cm, pH 3–11; GE Healthcare, Upsala, Sweden) were passively rehydrated (30 V, 12 h). This was followed by isoelectric focusing using an Ettan IPGphor IEF unit (GE Healthcare). Focusing was performed at 20°C and 50 μA per strip, according to the following sequence: 500 V for 1 h, 1000 V for 1 h, 8000 V for 3 h, 8000 V up to a total of 45,000 Vh. After the first dimension, the strips were equilibrated and separated on 12.5% (SDS-PAGE) gels using an Ettan Dalt Six device (GE Healthcare). The gels were scanned using a Typhoon 9400 scanner (GE Healthcare) with appropriate wavelengths and filters for Cy2, Cy3, and Cy5 dyes. Total protein (1 mg) was obtained from a pool of equal protein amounts for each sample. This was denatured in lysis buffer and mixed in a rehydration buffer. Gels were then stained by Colloidal Coomassie Blue.

One-dimensional analytical gel electrophoresis was performed using Immobiline Dry Strips (24 cm, pH 3-11; GE Healthcare, Sweden) and then the IPG strips were equilibrated, first with dithiothreitol (DTT) (15 min, RT, gentle stirring, 5 mM Tris–HCl, pH 8.8, 6M urea, 30% glycerol, 2% SDS 65 mM). Strips were then equilibrated for 15 min in the same solution containing 250 mM iodoacetamide (IAA). followed by second-dimension sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) performed on 12.5% fixed gels using an Ettan Dalt Six device (GE Healthcare, Sweden) were carried out as previously described by Alfadda et al. [77 (link)]. Further, the 2D-DIGE gels were scanned on the Typhoon 9410 scanner using excitation/emission wavelengths specific for Cy2 (488/520 nm), Cy3 (532/580 nm), and Cy5 (633/670 nm).

Tissue proteins were labeled according to the manufacturer´s instructions (GE Healthcare), and as described by Gil-Dones and Martin-Rojas [8 (link), 9 (link)]. In all experiments an internal standard was added containing equal amounts of each protein extract. The internal standard and protein extracts from one stenotic and one control valve were combined and run on a single gel (150 μg of total protein). The proteins were separated by isoelectric focusing in the first dimension and the strips were then equilibrated via two consecutive incubations in SDS-equilibration buffer (1.5 M Tris∙HCl [pH 8.8], 6 M Urea, 87% Glycerol and 2% SDS) plus dithiothreitol and iodoacetamide, respectively. Subsequently, the proteins were separated on 12% Acrylamide/Bisacrylamide gels using an EttanDalt Six device (GE Healthcare), which were then scanned using a Typhoon 9400 fluorescence gel scanner (GE Healthcare). Relative protein quantification of Aortic Stenosic (AS) and healthy valves was performed with DeCyder software v6.5 (GE Healthcare) and with the multivariate statistical module EDA (Extended data analysis). Only proteins with >1.5-fold differences in abundance were considered significant. A statistical analysis was then carried out to determine the changes in protein expression, with p values below 0.05 accepted as significant when the Student’s t test was applied.

50 μg of total protein per sample
was covalently labeled with a fluorophore, either Cy3 or Cy5, by adding
400 pmol of CyDyes (DIGE Fluor dyes, GE Healthcare, UK) in 1 μL
of dimethylmethanamide (DMF), and then incubating for 30 min on ice.
To terminate the labeling reaction, 1 μL of 10 mM lysine was
added to each sample. In addition, an equal amount of all samples
in the experiment was pooled as an internal standard and labeled with
Cy2. Two-dimensional analysis gel electrophoresis was performed as
described by Alfadda et al.^{20 (link)} Briefly,
1 mg of total protein from the pool was added to the rehydration buffer
(7 M urea, 2 M thiourea, 4% CHAPS, 0.006 g DTT, 2 μL of bromophenol
blue, 5 μL of IPG buffer (pI 3–11), 1× protease
inhibitor mix) applied to 5 Immobiline Dry Strips (24 cm, pH 3–11;
GE Healthcare, Sweden). Isoelectric focusing was performed at 50 μA
per strip using an Ettan IPGphor IEF unit (GE Healthcare, Sweden,
30 V, 12h, 20 °C). Soon after, the strips were equilibrated and
separated on 12.5% (SDS-PAGE) gels using an Ettan Dalt Six device
(GE Healthcare, Sweden). The gels were scanned with the appropriate
wavelengths (Cy2, 488/520 nm; Cy3, 32/580 nm; and Cy5, 633/670 nm)
and filters for the CyDyes dyes using a Typhoon 9400 scanner (GE Healthcare,
Chicago, IL, USA).

For the first dimension of separation, nine dry strips of Immobiline strips (measuring 24 cm and at a pH of 3–11; GE Healthcare, Danderyd, Sweden) were passively rehydrated (30 V; 12 h), followed by isoelectric focusing using an Ettan IPGphor IEF unit (GE Healthcare, Danderyd, Sweden). The focusing was performed at 20 °C, with 50 µA per strip. After the first dimension, the strips were equilibrated and separated on 12.5% SDS-PAGE gels using an Ettan Dalt Six device (GE Healthcare, Danderyd, Sweden). The gels were then scanned using a Sapphire Biomolecular Imager (Azure Bio systems, Dublin, OH, USA), and digitalization was performed using the image analysis software Sapphire Capture system (Azure Biosystems, Dublin, OH, USA). Preparative gels were prepared using the total protein (1 mg) obtained from a pool of equal protein amounts. The gels were then stained using Colloidal Coomassie Blue, as described previously [17 (link),30 (link),31 (link)].