The MAPs fraction is now ready for analysis by electrophoresis. All steps of purification are schematically shown in Figure 1. 3.4 Proteins analysis on SDS-Polyacrylamide gel electrophoresis 3.4.1 1D-SDS electrophoresis gel profile 1. Motors and MAPs fraction are loaded on a 6 to 18 % gradient electrophoresis gel on a vertical slab gel at 25mA/ gel at 4°C. 3. After migration (until the front reached the bottom of the gel), the gels are stained with Coomassie Blue ( see Note 12 ). Analysis of this gel is shown in Figure 2 ( see Note 13 ). 6
Gache et al 3.4.2 2D-SDS electrophoresis gel profile 1. Two-dimensional (2D) electrophoresis is performed with immobilised pH gradients for isoelectric focusing. Home-made linear 3-10.5 gradients are used (22) and prepared according to published procedures (23). IPG strips are cut with a paper cutter, and rehydrated in 7 M urea, 2 M thiourea, 4% CHAPS, 0.4% carrier ampholytes (3-10 range) and 5mM Tris cyanoethyl phosphine (Molecular Probes, Cat. No T6052) for 3-10.5 gradients (24). The protein sample is cup-loaded at the anode. Isoelectric focusing is carried out for a total of 60000 Vh ( see Note 14 ). 2. After focusing, the strips are equilibrated for 2 x 10 minutes in 6 M urea, 2% SDS, 125 mM Tris-HCl pH 7.5 containing either 50mM DTT (first equilibration step) or 150mM iodoacetamide (second equilibration step). The equilibrated strip is loaded on the top of a 10% polyacrylamide gel, and submitted to SDS PAGE (10% gel) at 12W/ gel (25). 3. After migration, the gels are stained with colloidal Coomassie Blue (26) ( see Note 15 ). Analysis of this gel is shown in Figure 3. 3.5 Mass spectrometry analysis of proteins resolved on SDS- electrophoresis gels 3.5.1 In-gel digestion of proteins bands 1. Coomassie Blue-stained bands (spots) of interest are excised from 1D or 2D gels and digested in-gel as described in (27, 28) ( see Note 16 ). 2. Briefly, gel pieces are cut in ca. 1 mm x 1mm cubes and dehydrated with acetonitrile. Proteins are reduced with 10 mM DTT in 100 mM ammonium bicarbonate at 56°C and alkylated with 55 mM iodoacetamide. After washing with 100 mM ammonium bicarbonate and dehydration with acetonitrile, a sufficient volume of digestion buffer (12.5 ng / l of trypsin in 40 mM NH 4 HCO 3 /10 % acetonitrile) is added to cover the gel pieces. Samples are first incubated 2h at 4°C, and the digestion is then performed overnight at 37°C, after addition of more buffer if necessary ( see Note 17 ). 3. After digestion, peptides are extracted, successively, with 50 l (equal to 1-2 times the volume of gel particles) of acetonitrile and 100 l of acetonitrile: 5% formic acid (50:50). The extracts are pooled together, dried down in a vacuum centrifuge and stored at -20°C ( see Note 18 ). 3.5.2. NanoLC MS/MS sequencing 1. Dried samples are re-dissolved in 15-25 L of 0.05 % trifluoroacetic acid (TFA) and 4 L are loaded onto the trap column in 0.05 % TFA at the flow rate of 20 L/min ( see Note 19 ). After 4 min of loading and washing, peptides are eluted and separated on the analytical column at the flow rate of 200 nL/min with the following gradient: from 5 to 20 % of solvent B in 20 min, 20 to 50 % B in 16 min, 50 to 100 % B in 5 min, 100 % B during 10 min, and back to 5 % B in 5 min. Solvent A: 95:5 H 2 O:acetonitrile (v/v) with 0.1 % formic acid (v/v); solvent B: 20:80 H 2 O:acetonitrile (v/v) with 0.1 % formic acid
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