Laemmli buffer 4x

Lipolytic activity of secreted CalB was determined using a p-nitrophenyl butyrate (pNPB)-based assay (2635–84–9, Merck, Darmstadt, Germany). The reaction buffer was as follows: 300 mM Tris-HCl pH 7 containing 4.83 mM p-NPB and 0.93% acetone. After pre-heating the buffer at 30°C, 900 μL of reaction buffer were mixed with 100 μL of culture supernatant. P-nitrophenol color development at 405 nm was monitored in a Specord 200 Plus spectrophotometer (Analytik Jena GmbH, Jena, Germany) at 30°C for 2 min. One activity unit was defined as the amount of enzyme needed to release 1 μmol p-nitrophenol · min^-1 under assay conditions. RSD was below 4%.
Further protein analyses of fed-batch supernatant samples were performed by SDS-PAGE. For sample preparation, 15 µL of supernatant were mixed with 5 µL of loading buffer (Laemmli buffer 4x + β-mercaptoethanol in 10:1 relation, Biorad, Hercules, CA, USA) and incubated at 95°C for 5 min. After cooling down, 15 µL were loaded into 4%–15% Mini-PROTEAN TGX Precast Protein Gels (Biorad, Hercules, CA, United States) and run at 120 V for about 80 min. The visualization and analyses of the gel was performed using Image Lab (Biorad, Hercules, CA, United States).

T. trichiura EE (10 μg/well) and FE (10 μg/well) were diluted in Laemmli buffer (4X) (Bio-Rad) (1:1), denatured at 100°C for 5 min and separated by one dimensional gel electrophoresis (1-DE) in Mini-Protean TGX precast acrylamide gels (4–15% gradient, 10 well comb, 50 μL/well) (Bio-Rad) under reducing conditions with 80–120 V in a Mini-PROTEAN Tetra System electrophoresis system (Bio-Rad) as previously described [32 (link)]. Samples were run simultaneously with molecular weight markers (4 μL) (Precision Plus Protein Dual Color Standards, Bio-Rad).
The gels were stained with Coomassie brilliant blue to analyze the protein patterns and the most prominent bands excised for proteomic analysis. Before staining, the gels were fixed (50% methanol and 10% glacial acetic acid) overnight with gentle agitation (solution changed once after 1 h). Gels were stained (0.1% Coomassie brilliant blue R-250, 50% methanol and, 10% glacial acetic acid) for 20 min with gentle shaking before destaining (40% methanol and 10% glacial acetic acid) with repeated changes of the solution until the gel background was clear. Gels were stored at 4°C in 5% glacial acetic acid.