Novex sharp pre stained protein standard

Recombinant FAE type A from A. niger (AnFaeA) was expressed in Pichia pastoris as described previously^{28 (link)}. For the synthesis of arabinose esters, a type B FAE from Myceliopththora thermophila ATCC 42464 (MtFae1a) was expressed in P. pastoris, as described previously^{20 (link)}. The enzymatic preparations were concentrated and exchanged for 100 mM MOPS-NaOH pH 6.0. Protein concentration was determined by the Pierce^TM BCA Protein Assay (ThermoFisher Scientific, USA). The FAE purity (%) of the enzyme preparations was determined by SDS-PAGE using a Novex Sharp pre-stained protein standard (Life Technologies, USA), followed by quantification using the JustTLC software (Sweday, Sweden).

Twenty-five μg of total protein from brain or 10 μg of total protein from cortical neurons in culture were used to assess protein expression by WB. Proteins obtained from control, NMDA or OGD samples following IP were prepared in WB loading buffer and loaded in Precast NuPAGE Midi 10% Bis-Tris (Life Technologies). MW markers (Magic Mark XP or Novex Sharp Pre-Stained Protein Standard; Life Technologies) were included in 10% Bis-Tris gels. For protein extraction addressed to detect phosphorylated proteins, we used a lysis buffer containing 20 mM Tris-Cl pH 7.5, 137 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 100 mM phenylmethylsulfonyl fluoride and Complete™ EDTA-free Protease Inhibitor Cocktail supplemented with 2 mM EDTA, 5 mM sodium orthovanadate and 50 mM sodium fluoride. The presence of the proteins of interest was detected and analyzed with an image generation and analysis system based on the Odyssey Near Infrared Fluorescence (Li-COR). When required, actin or tubulin were used as tissue sample loading controls and to normalize data in expression experiments. DAPK-1 band patterns obtained in WB using the two anti-DAPK1 antibodies (a polyclonal Ab against aa of the N-term DAPK1 molecule or a monoclonal clone DAPK-55 against the whole molecule) listed in the antibodies section were similar in recognizing both full-length and truncated DAPK1 forms.

GUS ELISA was performed essentially as described by Dugdale et al. [33 (link)]. For PAGE analysis, TSP (20 μg) was electrophoresed through a NuPAGE^® Novex^® 4–12% Bis–Tris Protein Gel (Life Technologies) at a constant voltage (200 V) for 55 min in NuPAGE^® MOPS SDS Running Buffer with NuPAGE^® Antioxidant (Life Technologies) according to manufacturer’s specifications. As a control, 0.3 μg of purified GUS protein (GUS Type VII-A; Sigma-Aldrich G7646) was loaded. Protein sizes were estimated using the Novex^® Sharp Pre-stained Protein Standard (Life Technologies). Following electrophoresis, the acrylamide gel was stained in Coomassie Brilliant Blue dye overnight (approximately 16 h) and destained in 15% ethanol/10% acetic acid.