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In the first step of the separation, 3 µl of the combined peptide mixture were loaded onto the PolySulfoethyl A strong cation exchange column (SCX) (0.32×50 mm, 5 µm) [26].
An aliquot of 7.5 µl of the mixed complexes were transferred into a new Eppendorf tube and mixed with an equal volume of sample buffer (0.1 M Tris-glycine buffer containing 20% glycerol and bromophenol blue) and 15 µl of the mixture were loaded on 8% native polyacrylamide gel electrophoresis (PAGE) without SDS as previously described [18].
150 μg protein of pooled, mixed samples and in addition 100 μg unlabelled protein mixture were loaded via anodic cup loading onto IPGstrips.
Samples of 200 μL of this mixture were loaded into a preparative reverse phase HPLC column to separate the treated enzyme (PrTX-III: HP-2) from HP-2.
After denaturation at 98 °C for 10 min and quenching on ice, 20 μl of the mixture were loaded onto a gel containing 0.8 × MDE (Cambrex Corporation, East Rutherford, NJ, USA) for exons 12 and 18 and 1 × MDE for exon 23, and 0 3% Glycerol (0% for exon 12 and 3% for exons 18 and 23).
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The mixture was loaded onto a gel filtration column (Superose6 10/300 GL, GE Healthcare).
Then, the mixture was loaded into a PTFE lined hydrothermal synthesis reactor.
The first-round PCR reaction mixture was loaded into the capillary and covered by oil.
The mixture was loaded onto a Superdex G200 column again to separate the Uch37-Rpn13C complex from excess Rpn13.
The hydrogel mixture was loaded to a surface-treated PDMS mold using a pipette or a syringe.
The mixture was loaded into a Teflon-lined autoclave of 50 ml capacity under magnetic stirring and then tightly closed.
Related(20)
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