Mini protean tgxtm 4 20 polyacrylamide precast sds page gels

Extracted crystals were prepared for analysis by SDS-PAGE by boiling with SDS-PAGE sample buffer containing β-mercaptoethanol. Samples were then run on Mini-PROTEAN^® TGX^TM 4–20% polyacrylamide precast SDS-PAGE gels (BioRad) and stained using Coomassie blue. Gels were scanned using a Gel Doc^TM XR+ system (BioRad), and molecular weight and relative quantity estimates for gel bands were calculated using Image Lab software version 5.0 (BioRad).
Each band was excised from the gel, subjected to tryptic digestion, and peptides analysed on a 4800 MALDI tandem Time-of-Flight Analyzer (MALDI TOF/TOF, Applied Biosystems, MA) at the Centre for Protein Research (University of Otago, Dunedin, New Zealand). The resulting peptide mass data were searched against the combined squid transcriptome assembly translated into all possible reading frames, using the Mascot search engine (http://www.matrixscience.com).

200 µl of each fraction eluted from FPLC was precipitated by adding 800 µl of acetone chilled to −20 °C, then centrifuged at 16,100 rcf for 10 min, the supernatant discarded and pellet left at 37 °C for 20 min to dry. The pellet was resuspended in 10 µl of loading buffer containing β-mercaptoethanol before being loaded onto the gel. Samples were run on Mini-PROTEAN TGXTM 4–20% polyacrylamide precast SDS-PAGE gels (BioRad) and stained using Coomassie blue. Gels were scanned using a Gel DocTM XR + system (BioRad), and molecular weight estimates for gel bands were calculated using Image Lab software version 5.0 (BioRad).