Roti blue

For separation of proteins in native agarose gels, recombinant VEGF (Life Technologies, Carlsbad, California), histones from calf thymus (Sigma-Aldrich, Steinheim, Germany) or catalase from bovine liver (Serva, Heidelberg, Germany) were each incubated with polySia (colominic acid from E. coli equal to polySia in mammals; GERBU, Heidelberg, Germany) in 50 mM Tris buffer for 1 h at 30 °C with agitation. The samples were loaded onto a 2% agarose gel (peqGOLD Universal Agarose, peqLab, Erlangen, Germany) and separated using 19.2 mM glycine in 25 mM Tris/HCl (pH 8.5) buffer. The electrophoresis was run at 80 V (constant voltage) for 3.5 h [32 (link)]. Thereafter, the gel was fixed in 45% methanol containing 7.5% acetic acid (v/v) overnight. Roti-blue (Roth, Karlsruhe, Germany) was used as Coomassie blue staining-dye according to manufacturer’s instructions.

Cells were harvested by centrifugation (6000 × g, 5 min, 4°C) and washed once with ZAP buffer. The cell pellet was resuspended in 1 ml ZAP buffer also containing 1 mM PMSF and disrupted by sonication. Cell debris were removed by centrifugation and the resulting supernatant was considered as total cellular protein extract. Aliquots of these samples were separated by standard sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Gels were either stained using the colloidal coomassie agent Roti^®-Blue (Roth, Germany) or transferred onto positively charged polyvinylidene fluoride (PVDF) membranes using a semi-dry transfer unit. Proteins of interest were immune-stained using polyclonal rabbit antisera recognizing DivIVA (Marston et al., 1998 (link)) or SecA2 (this work) as the primary antibody and anti-rabbit immunoglobuline G conjugated to horseradish peroxidase as the secondary one. The ECL chemiluminescence detection system (Thermo Scientific) was used for detection of the peroxidase conjugates on the PVDF membrane in a chemiluminescence imager (Vilber Lourmat). Protein identification by mass spectroscopy was performed as described recently (Halbedel et al., 2014 (link)).

The 2D protein separation was carried out as described earlier30 (link). The 2-DE gels were stained with Flamingo fluorescent gel stain (Bio-Rad, Hercules, CA, USA) following the manufacturer instructions. After staining, gels were scanned at 50 μm resolution on a Fuji FLA-5100 scanner. The digitalized images were analyzed using Delta 2D 3.4 (Decodon, Brunswick, Germany). For protein visualization, the 2-DE gels were additionally stained overnight with colloidal Coomassie blue, Roti-Blue (Roth, Karlsruhe, Germany).

The intracellular domain of DEP-1 (wild-type and D1241A) was cloned into the BamHI site of the E. coli expression vector pGEX-2TK (Pharmacia) as described [11 (link)]. Recombinant proteins were affinity-purified on glutathione Sepharose according to the manufacturer’s protocol, except that protein expression was induced in BL21 bacteria at 18°C, and fusion proteins were washed in 20 mM NaP pH = 8.0, 250 mM NaCl, 1% Triton-X. Approximately 50 μg of each DEP-1 fusion protein (wild-type/D1241A) and 100 μg of GST as a negative control were used in each binding reaction. To prepare N2 worm extracts, mixed-stage liquid cultures were cleaned by sucrose flotation, resuspended in lysis buffer (100 mM Tris pH = 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.5% NP-40, 1x protease inhibitor cocktail; Roche), shock frozen in liquid nitrogen, and homogenized in a swing mill (MM300; Retsch). Thawed worm extract was then centrifuged for 10 min at 4°C and 10,000g to remove insoluble components. About 2.5 mg of total protein extract was used in each reaction. Binding was performed at 4°C overnight, followed by three washes with lysis buffer. Bound proteins were eluted by boiling the beads for 5 min in 30 μl Laemmli buffer and separated on a 4–15% linear gradient SDS-gel (Biorad Nr. 161–1104), followed by colloidal Coomassie blue staining according to the manufacturer’s protocol (Roti-Blue; Roth).

For image analysis, 2-DE gels were fixed in a solution containing 50% methanol and 12% acetic acid overnight and fluorescent stained with Flamingo fluorescent gel stain (Bio-Rad, Hercules, CA, USA) for minimum 5 h. Thereafter, gels were scanned at 50 μm resolution on a Fuji FLA-5100 scanner using the Image Reader Software (Fuji). The digitalized images were analyzed using Delta 2D 4.3 (Decodon, Braunschweig, Germany). For protein identification, 2-DE gels were additionally stained with colloidal Coomassie blue, Roti-Blue (Roth, Karlsruhe, Germany) overnight.

Tandem affinity-purified Dhr1-FTpA was loaded on a 15–40% (w/v) linear sucrose gradient containing 100 mM NaCl, 50 mM Tris-HCl pH 7.5, 1.5 mM MgCl₂, 0.01% Igepal, 1 mM DTT. Gradients were centrifuged at 129,300×g (4 °C) in a SW40 rotor (Beckman Coulter) for 16 h. Respective fractions were collected and TCA-precipitated (final concentration 12.5%), the pellets were washed with aceton and subsequently resuspended in SDS-sample buffer. Proteins were analyzed utilizing 4–12% gradient polyacrylamide gels (NuPAGE, Invitrogen) and stained with colloidal Coomassie (Roti-Blue, Roth).