4 MATERIALS AND METHODS
4.4 Methods for protein analysis
Protein preparation from H. salinarum cells
Preparation of membrane and cytosolic protein fractions for use in immunoblots and for MS analysis
Membrane and cytosolic fractions of halobacterial protein were prepared under high salt conditions (1.3 M) as follows: 450 ODml of cells from a 350 ml culture with an OD600 of 1.4
were spun down, and resuspended in 20 ml of 30 % basal salt medium (30% complex medium without peptone) additionally containing 10 µg/ml PMSF, 20 mM HEPES pH 7.5 and 100 µg/ml DNase I. After sonication at 0 - 10°C (2 x 1 min, Branson sonifier 450, 1/2" disruptor horn, 30% duty cycle, level 6) the lysate was centrifuged at 6,300·g and 20°C for 20 min to remove unlysed cells and allow for DNase I action. The membrane protein fraction was obtained as a pellet after centrifugation (60Ti-rotor, Beckman, 200,000·g, 90 min, 4°C). After removing the supernatant the pellet was resuspended in 20 ml of the mentioned buffer and centrifuged as before. It was finally resuspended in 1 ml of protein resuspension buffer (20 mM HEPES pH 7.5, containing 10 µg/ml of PMSF and 1 mM EDTA). To avoid contamination of the cytosolic protein fraction with the pellet only the upper 7 ml of the supernatant of the first ultracentrifugation were carefully collected, the rest was discarded. After protein precipitation by
adding 6 volumes of acetone (-20°C) to the saved supernatant, followed by two washing steps with 40 ml ice cold 50% acetone to remove the salt, the precipitate was pelleted, dried and resuspended in 1 ml of protein resuspension buffer. Aliquots were stored at -70°C.
4.4.2
Determination of protein concentration
Protein concentrations of membrane and cytosolic fractions were determined with the BCA protein assay reagent kit (Pierce, Rockford, IL, USA) according to the manufacturer's recommendations. The method is based on the biuret reaction of Cu2+ with proteins in alkaline
solution to form Cu+, followed by the sensitive and selective colorimetric detection of Cu+ via its
reaction with bicinchoninic acid (BCA) to form a blue complex with an absorption maximum at 562 nm. In brief, the assay was performed in a microplate in which different dilutions of a BSA standard (2 mg/ml) and different dilutions of protein samples of unknown concentrations were separately mixed with the reagent mix and then incubated at 60°C for 30 min. After the determination of absorption values at 490 nm in a Wallac 1420 multiwell plate reader (Perkin Elmer, Boston), a standard curve was generated with the BSA dilutions and used to determine the concentrations of the unknown samples.
4.4.3
SDS PAGE
Proteins from the halobacterial membrane or cytosolic fractions, which had been stored at -70° C, were incubated for 20 min at 40°C in 1 x SDS sample buffer prior to their electrophoretic separation (15 µg total protein per lane) in a discontinuous SDS PAGE according to Laemmli (1970). The acrylamide content was 3.9% for the stacking gel and 8% for the separating gel. The electrophoresis runs were performed in SE250 Mighty Small II miniature vertical slab gel units (Hoefer Scientific Instruments, San Francisco, CA, USA) under constant cooling with tap water. The 8 cm x 7 cm x 0.75 mm gels were subjected to currents of 5 mA per gel until the front of bromophenol blue reached the separating gel and ≤ 15 mA per gel for the remaining time at a maximum voltage setting of 150 V. The total electrophoresis time amounted to approx. 3.5 hours. In each case the BenchMark protein ladder (Invitrogen, Karlsruhe, Germany) was used as a molecular weight standard. In addition to the components listed below, the separating gel optionally also contained 6 M urea to reduce the number of bands detected on immunoblots.
The separated proteins were finally either analyzed immunochemically or via in-gel digestion followed by MS analysis of the generated peptides. To visualize protein bands, the gels were incubated with Coomassie stain for 15 min, followed by destaining in Destain-1 for 10 min and in Destain-2 for 30 min or up to several hours. Staining and destaining solutions were heated in
a microwave to approx. 40 - 50°C before the addition of the gels. To avoid contaminations in the subsequent MS analyses, these solutions were prepared from chemicals of the purity grade p.a. and from ultrapure water (18.2 MΩcm) generated with the Milli-Q PF Plus system (Millipore Corporation, Billerica, MA, USA).
3.9% Stacking gel (5 ml for 3 minigels)
3.9% acrylamide (aa) / bisacrylamide (baa)-mix 650 µl 30% aa / 0.8% baa-mix
125 mM Tris/HCl pH 6.8 630 µl 1 M (8x) Tris/HCl pH 6.8
0.1% (w/v) SDS 50µl 10% (w/v) SDS (350 mM)
ad 5 ml H2O 3.7 ml H2O
0.2% TEMED 10 µl TEMED
0.1% (w/v) APS (add at last!) 50 µl 10% (w/v) APS
Addition of 20 µl of 2% bromophenol blue (in ethanol) slightly stains the gel and facilitates loading of the samples.
8% Separating gel (10 ml for 2 minigels)
8% acrylamide (aa) / bisacrylamide (baa)-mix 2.7 ml 30% aa / 0.8% baa-mix
375 mM Tris/HCl pH 8.8 2.5 ml 1.5 M (4x) Tris/HCl pH 8.8
0.1% (w/v) SDS 100µl 10% (w/v) SDS (350 mM)
ad 10 ml H2O 4.7 ml H2O
0.2% TEMED 20 µl TEMED
0.04% (w/v) APS (to be added at last!) 40 µl 10% (w/v) APS
5 x SDS electrophoresis buffer (used as 1 x SDS electrophoresis buffer)
125 mM Tris base 15.1 g Tris base
960 mM glycine 72 g glycine
0.5% (w/v) SDS 5 g SDS
ad 1000 ml H2O ~ 1 l H2O
For use at the anode, the buffering capacity should be increased by addition of 7 g Tris base per liter 5 x buffer, resulting in a pH of approximately 8.8. 5 x SDS sample buffer (10 ml) 300 mM Tris/HCl pH 6.8 3 ml 1 M Tris/HCl pH 6.8 9% (w/v) dithiothreitol (580 mM) 900 mg dithiothreitol 50% (v/v) glycerol 5.9 ml 87% glycerol 12% (w/v) SDS 1.2 g SDS
0.03% bromophenol blue 150 µl 2% bromophenol blue (in 20%
ethanol)
The sample buffer was stored in aliquots at -20°C. When gel electrophoresis was followed by MS analysis or immunoblotting, protein samples were dissolved in 1 x SDS sample buffer, which additionally contained freshly added 1.5% (v/v) 2-mercaptoethanol, to ensure complete reduction of disulfide bonds.
Coomassie stain (500 ml)
0.1% Coomassie brilliant blue R250 0.5 g Coomassie brilliant blue R250
40% (v/v) ethanol 200 ml ethanol
10% (v/v) acetic acid 50 ml acetic acid
ad 500 ml H2O 250 ml H2O
Destain-1
Like Coomassie stain but without the Coomassie brilliant blue R250.
Destain-2