filled with 40 mL column buffer in the near chamber and 40 mL column buffer complemented by 0.896 g KCl in the front chamber. The protein solution was added on the column. After the sample had entered the column bed, it was washed with two column volumes of column buffer (slow flow velocity). Sample collection began when the collected volume had reached the dead volume of the column. Therefore, the salt gradient was started and the column volume was kept constant by regulation of inflow and outflow. The sample volume was collected in 1.5 mL Eppendorf cups and protein concentration was monitored by UV-Vis spectrometry (NanoDrop ND-1000, ThermoScientific Technologies, Inc., Wilmington, USA).
As an additional quality control, an 8 % SDS-gel was performed prior to the final purification step. Samples collected during the purification process and samples taken from protein frac-tions after DEAE column were mixed with sample buffer – 10µl sample (in case of high protein concentration 5µl protein solution and 5µl MilliQ water), 12.5µl sample buffer, 22.5 ml MilliQ water and 5µl 1 M DTT. Prepared samples were denatured at 70◦C for 10 min and loaded on the gel, a protein marker was added to the first well. The gel was placed in an electrophoresis system (VWR/CBS, CBDCX-700, Darmstadt, Germany) and performed for 75 min at 100 mA (for two gels 200 mA). Afterwards, the gel was stained using InstantBlueTMfor 15 to 60 min.
The fractions with the highest protein concentration after DEAE column were pooled for a cation exchange chromatography. This second chromatography was performed in analogy to the anion exchange chromatography with the following changes. The column was filled with CM-Sepharose as column material and 40 mL column buffer complemented by 1.792 g KCl, were filled in the front chamber. After the sample was added to the column the salt gradient was started directly. The protein concentration of the eluate was monitored by measuring the absorption at 280 nm using the NanoDrop. Fractions with a high protein concentration were pooled. Lastly, MAC was added until a final concentration of 10 mM was reached. Aliquots of the protein solution were stored at -80◦C . The protein should, according to Harald Herrmann (DKFZ, Heidelberg, Germany), not be used for longer than three years after production. Thaw-ing and re-freezThaw-ing should be avoided as much as possible.
3.2 Labeling of Vimentin
For fluorescence microscopy vimentin had to be labeled with a fluorescent dye, in this case ATTO647N. OT stretching measurements of vimentin requested coupling to a bead. In most of the experiments a biotin streptavidin reaction, with biotin labeled vimentin, was used for coupling. The very specific sulfhydryl-reactive crosslinker chemistry was used to ensure that only the cysteine at the tail domain was labeled and the interaction of the label with the α
-helical rod domain and the hierarchical formation of the vimentin filament was minimal.
Figure 3.1:Reaction scheme of the maleimide cysteine coupling reaction.
3.2.1 Labeling with Fluorescent Dyes
The experimental procedure for vimentin labeling via maleimide chemistry was adopted from references [4, 5]. 500µl vimentin (concentration approx. 2.7 g/L) was placed into a 50 kDa dial-ysis tubing (Spectra/Por®7, dialdial-ysis membrane made of regenerated cellulose, MWCO 50000, E883.1, Carl-Roth GmbH, Karlsruhe, Germany) and dialyzed into labeling buffer (50 mM NaH2PO4, 50 mM Na2HPO4, 5 M urea, pH 7.0) at 10◦C over night.
After dialysis, vimentin solution was placed in an 1.5 mL reaction tube and the concentration was adjusted to approximately 1 g/L by dilution with labeling buffer (monitored by UV-vis spec-troscopy). 1 mL of this solution was filled into in a new 1.5 mL reaction tube. 20µl of 10 mM ATTO647N-maleimide, dissolved in DMSO, were added stepwise in 5µl portions. Each addition of ATTO647N was followed by an incubation step of 5 min, where the tube was placed on a shaker. Subsequently, the sample was incubated for 120 min at room temperature to complete the labeling reaction. Lastly 100µl 1 M L-cysteine were added and the mixture was incubating for another 1 h at room temperature to ensure that the remaining maleimide reacted and did not bind unspecific to vimentin.
Free dye and labeled protein were separated by size-exclusion chromatography, using a 300 mm Bio-Gel P polyacrylamide gel column with a bed volume of 24 mL. Column material (BioGel P30) was hydrated in labeling buffer at room temperature over night and the column was prepared as recommended in the instructions manual. The protein-label solution was placed on top of the column and was allowed to enter the bed completely. Labeling buffer was used to flush the column and fractions with labeled vimentin were collected in about 250µl aliquots. Protein and dye concentration were monitored by UV-vis spectroscopy. Fractions with the highest protein concentration were pooled. The final protein and dye concentrations was measured (UV-vis spectroscopy) and the labeling ratio was calculated. Lastly the labeled vi-mentin was dialyzed into storage buffer and stored at -80◦C in aliquots of about 50 to 100µl.
3.2. Labeling of Vimentin 39
Figure 3.2:Excitation and emission spectrum of ATTO647N. Ideal wavelength for excitation is λex = 646nm and the maximum emission wavelength λem = 664nm. Image from https://www.atto-tec.com/attotecshop/product_info.php?info=p114_atto-647n.html.
Different from unlabeled vimentin, storage buffer for labeled vimentin only contained 2 mM phosphate buffer, pH 7.5 and 8 M urea. Aliquots were used up within two years.
3.2.2 Labeling with Biotin
The procedure for labeling with maleimide-biotin was mostly identical to the labeling with fluo-rescent dyes except for the size exclusion chromatography. Separation of labeled vimentin and free maleimide-biotin was performed using three prepacked and disposable PD MidiTrap G-25 columns containing 3.5 mL Sephadex G-25 resin (GE Healthcare Europe GmbH, Freiburg, Ger-many) which were equilibrated with labeling buffer. After labeling and addition of cysteine, the sample was placed on the first column and eluted by adding 1.5 mL labeling buffer, yielding one fraction of 1.5 mL. Half of this fraction was added to the other two columns, each. To completely fill the columns, 250 mL labeling buffer was added to each of them after the protein solution had entered the bed. Subsequently biotin-labeled vimentin was eluted using 1.5 mL labeling buffer on both columns. The collected flow-through was pooled and, after dialysis into storage buffer (as dye-labeled vimentin), aliquoted into 100µl portions for storage at -80◦C . Different from dye-labeled vimentin it was not straight forward to measure the protein and biotin con-centration in the sample after labeling, even though a UV tracker biotin was used. Therefore, biotin-labeled vimentin was only added to an amount that ensured that the total amount of biotin-labeled vimentin in the sample did not exceed 10 %. The volumes that had to be mixed were calculated due to the vimentin concentration of the unlabeled vimentin and the vimentin prior to the labeling reaction. Because of the two MidiTRap column steps the sample is diluted
by a factor of three and the vimentin concentration not higher than 0.3 g/L anymore.