Protein-biochemistry standard methods

To quantitatively determine the concentration of proteins, the BCA^TM Protein Assay Kit (Pierce) was used according to the manufacturer’s instructions. It is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantification of total protein. Cu²⁺ is reduced to Cu¹⁺ by protein in an alkaline medium (Biuret Reaction). The BCA chelates Cu¹⁺ ions forming purple-coloured complexes with an absorption maximum at 562 nm (Smith et al., 1985).

Another method that was sometimes employed to determine the protein contents was Bradford protein assay. It involves the binding of Coomassie Brilliant Blue G-250 dye to proteins . The dye exists in three forms: cationic (red), neutral (green), and anionic (blue). In acidic conditions, the dye is primarily in the cationic form with absorption at 470 nm. When the dye binds to a protein, it is changed to the blue colour, and the absorption changes to 595 nm. Using a protein standard (such as BSA in known concentration), it is possible to estimate the protein concentration of the sample solution.

2.2.2 PCI (Phenol-Chloroform-Isoamylalcohol) extraction

The Phenol-Chloroform-Isoamlyalcohol extraction (PCI-extraction) is a method that is used for separating proteins from nucleic acids. The sample solution was supplied with the same volume of PCI-solution and thoroughly mixed. The mixed suspension was centrifuged for 5 min at room temperature at 13000 rpm to separate the aqueous and organic phases. The two phases were separated as the upper aqueous phase and the lower organic phase. Carefully remove the upper phase in a separate microfuge without mixing the two phases. The nucleic acids in the upper aqueous phase were precipitated by the addition of 1/10 volume of a 3 M NaOAc pH 5.2 and 2.5 volume of 100% ethanol. To increase precipitation of RNAs, 15-30 µg of glycoblue was added as a carrier. The proteins in the lower organic phase were precipitated with 2.5 to 3 vol. of cold 100% ethanol. Nucleic acids or proteins were precipitated overnight at 20°C or one hour at -80°C. Subsequently, they were pelleted for 20-30 min at 4°C, 13000 rpm. The pellet was washed

with 70% (v/v) cold ethanol and pelleted again. The recovered pellet was dried for 2-3 min under vacuum and resuspended in an appropriate buffer or RNAase free Milli-Q H₂O.

2.2.3 Denaturing polyacrylamide gel electrophoresis (SDS-PAGE)

Proteins were separated by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) adapted from (Laemmli, 1970) or by commercially available pre-cast NuPAGE^TM Bis-Tris gels (4-12%), for MS analyses. Protein samples were dissolved in 1x SDS loading dye and proteins were denatured for 5 min at 95°C and loaded onto a high TEMED (0.33% (v/v)) polyacrylamide gel (PAA) (acrylamide (AA):bisacrylamide (BA) = 37.5:1). 0.033% (w/v) APS was used for polymerization. The separating gel used varied from 12% to 8%. The stacking gel was 5% PAA.

Gels were run in a vertical gel running unit in a 1x SDS-PAGE running buffer until the bromophenol blue dye reached the bottom. Electrophoresis was done at 200V for 45-50 min using Bio-Rad mini gel systems. The protein bands were visualized either by Coomassie or by silver staining.

5% Stacking gel 8% separating gel 4x stacking gel buffer 375.0 µl 4x separating gel buffer 1250.0 µl

Rotiphorese Gel 30 250.5 µl Rotiphorese Gel 30 1332.5 µl H2O 874.5 µl H2O 2417.5 µl APS 10.0 µl APS 16.75 µl TEMED 1.0 µl TEMED 16.75µl For commercial gels, the manufacturer’s instructions were obeyed.

2.2.4 Coomassie staining

This protein staining technique exploits the Coomassie Brilliant Blue G-250, which is a triphenylmethane dye forming stable noncovalent complexes with proteins. Coomassie staining of protein gels was basically performed as described by Sambrook, Russell (Sambrook et al., 1989). Coomassie staining of proteins was performed after fixation of SDS denaturing gels in 40% (v/v)

Methanol / 10 % (v/v) acetic acid for 30 min at room temperature on a shaker. After rehydration of the gel in Milli-Q H₂O, the pre-heated staining solution was added on top of the gel.

Incubation was continued for at least 1 h at room temperature with gentle shaking. After that, the gel was destained with water with mild shaking until protein bands were clearly visible. Finally,

the gel was scanned, and individual lanes were analysed by mass spectrometry (Research group Mass Spectrometry -MPI-BPC, Göttingen)

2.2.5 Silver staining of protein gels

Silver staining of protein gel was mainly performed as described by Blum et al. (Blum et al., 1987). Before staining, all gels were fixed using the fixing solution of 40% (v/v) methanol and 10% (v/v) acetic acid for at least 30 min. During the entire process, all the solutions were kept at least 10 times the gel volume, and all steps were performed on a shaker. After fixation, the gel was washed twice with 50% ethanol and once with 30% ethanol for 20 min each. The gel was then submerged into a 0.8 mM Na₂S₂O₃ solution for precisely 60 seconds. Immediately afterward, the gel was washed three times shortly with ddH₂O to remove the thiosulfate from the surface of the gels. After that, the gel was incubated with 0.012 M AgNO₃, 0.026% formaldehyde solution for 30 min. After carefully removing the staining solution, the gel was washed at least three times with ddH₂O before applying the developing solution containing 0.56 M Na₂CO₃, 0.0185%

formaldehyde, 16 µM Na₂S₂O₃. Developing was stopped with a solution of 40% methanol, 10%

acetic acid. The gel was then transferred to a Whatman paper and dried under vacuum at 80°C for 45 min.

2.2.6 Western blot analysis

After separating proteins by SDS-PAGE, proteins were transferred to a nitrocellulose membrane by the wet-blotting procedure, according to Burnette et al. (Burnette, 1981). The transfer was carried out for 2 h at 70 V and 4°C in 1x western blotting buffer (1x transfer buffer). Membranes were washed with ddH₂O, (stained with ‘Ponceau S’ if necessary) and blocked for 2 hr at room temperature (RT) or overnight at 4°C in 1x TBS, 0.1% (v/v) Tween-20, 5% (w/v) milk powder.

Immunoblotting was carried out for 1 hr at RT or overnight at 4°C with primary antibodies specified according to standard protocols (dilutions as per supplier’s protocol). Horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies were used as the secondary antibody (1 hr at RT, dilution 1:50000), and proteins were detected by enhanced chemiluminescence using an ECL kit according to the manufacturer’s instructions. Where TAP tag detection was required, the Peroxidase-antiperoxidase complex (PAP) was used to detect Protein A of the TAP tag, and no secondary antibody was required.