3.13.1 Preparation of yeast cell lysate
After yeast cells were grown to log phase and treated as indicated for each experiment they were transferred in a 2 ml screw-cap tube or a 15 ml falcon tube. Cells were lysed with the same amount of glass beads and lysis buffer, which was chosen according to assay requirements and supplemented with protease inhibitor (complete EDTA-free, Roche), phosphatase inhibitor and if applicable RiboLock (Thermo) to avoid RNA degradation. Homogenization and rupture of cells was performed three times using the FastPrep-24 machine at 4.5 m/s for 2 ml tubes and at 5.5 m/s for 15 ml tubes for 20 seconds. This was followed by centrifugation at 4,000 g for 5 min and at 21,000 g for 10 min resulting in the cleared lysate directly used for experiments.
3.13.2 Protein-protein and protein-RNA co-immunoprecipitation
Co-immunoprecipitations (IPs) were utilized to analyze interactions between proteins or proteins and their bound RNAs and performed as described earlier (Gross et al., 2007;
Hackmann et al., 2014). For most analysis 10 µl slurry of GFP-Trap beads (Chromotek) with covalently bound anti-GFP antibodies were used to purify GFP-tagged proteins. Precipitation of other proteins was performed by coupling 20 µg of the specific antibody (anti-myc,
56 anti-Mex67) with 20 µl slurry of IgG-sepharose beads. Beads were washed 4 times with the corresponding buffer before use.
PBSKMT buffer pH 7.5 1x PBS pH 7.5 3 mM KCl 2.5 mM MgCl2
0.5 % (v/v) Triton-X-100 RNA-IP buffer pH 7.5 25 mM Tris/HCl pH 7.5
100 mM KCl
0.2 % (v/v) Triton-X-100 0.2 mM PMSF
5 mM DTT
RiboLock, Protease Inhibitor
Commonly 400 ml cell culture was used, treated as indicated and pelleted. Cells were lysed in PBSMT buffer for protein-protein IPs and in RNA-IP buffer for protein-RNA IPs as described above. To control and compare protein contents of each sample 50 µl lysate were mixed with the same amount 2x SDS loading buffer, boiled 5 min at 95 °C and stored at -20 °C to be used as input control. For RNA Co-IPs 100 µl were taken and immediately frozen in liquid nitrogen as total RNA sample. Lysates and antibody-coupled beads were combined and if applicable additional substances (RiboLock, phosphatase inhibitor) added. Where indicated, lysates were treated with 0.2 mg/ml RNase A to degrade single-stranded RNAs. For most precipitations samples were incubated 3-4 h at 4 °C with overhead rotation. Immunoprecipitation of Hsf1-GFP was performed for 20 min at 25 °C for the untreated cells and at 37 °C for cells that were prior heat stressed at 42 °C. Incubation of the stressed lysate was not performed at 42 °C because after 20 min lysates started to get cloudy - potentially because proteins started to denature and precipitate. After incubation beads were washed a minimum of six times with 1 ml of the corresponding buffer with centrifugation between each washing at 400 g for 2 min at 4 °C. For reduction of unspecific signals beads were transferred in a new Eppendorf tube before the last washing step followed by elution in 35 µl 2x SDS loading dye, boiling at 95 °C for 5 min and storage at –20 °C until use. In RNA Co-IP applications beads were split in two portions – one for control of protein precipitation in western blot, one for isolation of co-precipitated RNA as described in 3.10.1.
Materials and Methods
57 3.13.3 SDS-acrylamide gel-electrophoresis (SDS-PAGE)
Total cell lysates or proteins that were purified via co-immunoprecipitation were separated by size and analyzed by denaturing SDS polyacrylamide gel-electrophoresis (SDS-PAGE) essentially described in (Garfin, 2009). A Tris-glycine buffer and denaturing system (Laemmli, 1970) were used together with an SDS-gel composed of 5 % stacking gel and 10 % resolving gel.
Staking gel (5 %) 16.7 % (v/v) Rotiphorese Gel 30 acrylamide mix 125 mM Tris/HCl pH 6.8
0.1 % (w/v) SDS
0.1 % (w/v) Ammoniumpersulfate (APS)
0.1 % (v/v) Tetramethylethylendiamin (TEMED) Resolving gel (10 %) 33.3 % (v/v) Rotiphorese Gel 30
375 mM Tris/HCl pH 8.0 0.1 % (w/v) SDS
0.1 % (w/v) APS 0.1 % (v/v) TEMED
Running buffer 25 mM Tris
192 mM Glycine 0.1 % (w/v) SDS
2x SDS-loading buffer 125 mM Tris/HCl pH 6.8 25 %(v/v) Glycerol 2 % (w/v) SDS
5 % (v/v) β-Mercaptoethanol Bromophenol blue
Gel components were mixed and polymerization initiated by addition of APS and TEMED.
Samples were mixed with SDS-loading buffer, boiled at 95 °C for 5 min and centrifuged at 22,000 g for 1 min. A size standard (10 µl unstained or 3 µl prestained marker) was loaded together with the samples and electrophoresis performed at 25 mA to let samples run into the staking gel, followed by 40 mA for resolving gel or at 5-7 mA if the gel was run overnight.
3.13.4 Coomassie staining of SDS-gels
Coomassie Brilliant blue was used to stain proteins in SDS-polyacrylamide gels as in general described earlier (Neuhoff et al., 1985). This method was mainly used to control expression of recombinant proteins.
58 Staining solution 50 % (v/v) Methanol
10 % (v/v) Acetic acid
0.25 % (w/v) Coomassie Brilliant Blue R250 Destaining solution 5 % (v/v) Methanol
10 % (v/v) Acetic acid
Gels were incubated in staining solution for 15 min, followed by destaining with destaining solution until bands became visible. Heating in a microwave and tissues that bound color particles accelerated destaining.
3.13.5 Western blot analysis
To detect proteins with specific antibodies, samples separated by SDS-PAGE were transferred on a nitrocellulose membrane using Western blot. The method described in (Towbin et al., 1979) was modified as follows.
Blotting buffer 25 mM Tris base
192 mM Glycine
20 % (v/v) Methanol (freshly added) Ponceau S solution 5 % (v/v) Acetic acid
0.2 % (w/v) Ponceau S
TBST pH 7.4 50 mM Tris pH 7.4
150 mM NaCl
0.1 % (v/v) Tween 20
A semi-dry blotting system was used to transfer the proteins on the membrane. The Anode was wetted with blotting buffer, then one layer Whatman paper in size of the gel was put on the anode followed by the membrane (Protran 0.45 µm), then the gel and finally another layer of Whatman paper. All components were equilibrated in blotting buffer and air bubbles removed before blotting for 1.5-2 h depending on the size of the proteins of interest. The size of the membrane defined the amperage (1.5 mA/cm2). After transfer proteins on the membrane were stained with Ponceau S solution. Washing with water made separated proteins as well as marker protein bands visible, which allowed marking of the size standard and cutting the membrane.
Unspecific protein binding sites were blocked with 5 % milk powder in TBST for 1 h.
Afterwards, primary antibodies (see 3.4) in 2 % milk powder/TBST were applied for 2 h at room temperature or overnight at 4 °C. Membranes were washed three times with TBST before incubation with secondary HRP-coupled antibody in 2 % milk powder/TBST for 2 h at room
Materials and Methods
59 temperature. This was followed by two washing steps in TBST and one in TBS (TBST lacking Tween 20) before the 1:1 mixed detection substrate was added and signals were detected using the Fusion-SL chemoluminescence detection system.