Biochemistry

3.2.2.1 Preparation of cleared cellular lysate

Cleared cellular lysates (CCL) were prepared from resting or stimulated human cell lines or primary B cells to analyse proteins by western blotting. Therefore, 1x10⁶ cells per 20 μl NP40-lysis buffer were lysed in 1.5 microcentrifuge tube for 30 min on ice. Afterwards, the cell lysates were centrifuged for 10 min, at 4 °C and 16000 x g. The resulting CCL was transferred into a new 1.5 microcentrifuge tube and the cell debris was discarded. In the end, the CCL was mixed with 3x Laemmli buffer, heated for 5 min at 95 °C and stored at -20 °C.

NP40-lysis buffer: 0.1375 M NaCl

0.05 M Tris/HCl (pH 7.8) 1 mM sodium-ortho-vanadate 0.5 mM EDTA

10 % Glycerol

1 % NP40

+ Protease inhibitor (Sigma P2714) in _ddH₂O

38 were resuspended in 1 ml FCS-free RPMI 1640 medium (R0) with a final cell number of 3x10⁷ DG75 cells/ml. After the transfer of cells to 1.5 ml microcentrifuge tubes, they were incubated for 30 min at 30 °C to reduce pre-stimulatory effects of the FCS containing RPMI culture medium. Stimulation was done using 20 µg/ml anti-human IgM F(ab’)₂ fragments or 10 µg/ml anti-human CD8 antibodies for desired time points. Subsequently, DG75 cells were spun down in a microcentrifuge, the medium was discarded and cells were lysed in 200 μl NP40-lysis buffer per 1x10⁷ DG75 cells on ice for 15-25 min. Then, cell debris was removed by centrifugation (16000 x g, 4 °C, 10 min) and the cleared cellular lysate (CCL) was transferred into a new 1.5 ml microcentrifuge tube. Afterwards, 30 μl of the CCL was mixed with 3x Leammli buffer and incubated for 5 min at 95 °C for denaturation of proteins. The remaining CCL was used for affinity purification experiments. pGEX-4T1 vector (GE Healthcare). The resulting plasmid was transformed into the E.coli strain BL21 and plated on a LB-agar plate (37 °C, overnight). Next, an overnight culture was inoculated using one E.coli colony and 4 ml LB-medium containing ampicillin (37° C, shaking). The following day, the overnight culture was used to inoculate 200-300 ml YT-medium containing ampicillin (37 °C, shaking). The expression of the GST-fusion protein

39 was induced at OD 0.6 with 100 μM IPTG at 37 °C shaking for 4 h. In the case of low yield of GST-fusion protein, the temperature was reduced to 25 °C or 30 °C during protein expression. Afterwards, the bacteria were harvested in 50 ml tubes by centrifugation (3000 x g, 15 min, 4 °C) and the obtained bacteria pellet was stored at -80 °C.

3.2.2.4 Preparation of recombinant GST-fusion proteins

One bacteria pellet was resuspended in 20 ml cold 1x PBS and lysed by ultrasonification on ice (3x30 s, 1 cycle, 50 %). To support the lysis of bacteria, 200 μl 10 % Triton-X-100 were added, followed by an incubation for 10-15 min on ice. Next, the bacteria cell debris was removed by centrifugation (3000 x g, 20 min, 4 °C). The supernatant was used for GST-fusion protein purification. For that purpose, 200 μl of gluthatione-sepharose beads were added to the supernatant, followed by a 2-3 h incubation at 4 °C on a rotator.

Afterwards, beads were pelleted by centrifugation (400 x g, 5 min, 4 °C), the supernatant discarded and the beads transferred into a 1.5 ml microcentrifuge tube, followed by three washing steps with 1 ml cold 1x PBS (300 x g, 3 min, 4 °C). In the end, the beads were resuspended in 250 μl cold 1x PBS and stored at 4 °C.

3.2.2.5 Elution of GST-fusion proteins

200 µl of sepharose beads binding the desired GST-fusion protein were mixed with 100 µl reduced L-Glutahione (10 mM in 1x PBS) in a 1.5 ml microcentrifuge tube. The mixture was incubated for 10 min on a rotator at RT followed by a centrifugation step (500 x g, 5 min, RT). The GST-fusion protein containing supernatant was transferred to a new 1.5 microcentrifuge tube and stored at 4 °C, whereas the sepharose beads were mixed again with 100 µl reduced L-Glutahtione. In total, three elution cycles were performed. The preparation (3.2.2.4) as well as the elution of GST-fusion proteins were controlled by SDS-PAGE followed by Coomassie staining. For Coomassie staining, SDS-gels were incubated for 15 min at RT on a shaker. Afterwards, the Coomassie solution was replaced with tap water to de-stain the SDS-gel overnight.

Coomassie staining solution: 0.1 % Coomassie Brilliant Blue R-250 40 % MeOH

10 % acetic acid

40 3.2.2.6 Affinity purification (AP)

Affinity purifications were perfomed in three different ways using either GST-fusion proteins, synthetic peptides or antibodies. For affinity purification with GST-fusion proteins, 15 μg of the desired GST-fusion protein was added to the CCL and incubated for 4 h rotating at 4 °C. In the case of synthetic peptides, 2 μM of biotinylated peptides were added and incubated for 1 h rotating at 4 °C. Afterwards, 25 μl Streptavidin-sepharose beads were added, followed by 1 h incubation at 4 °C on a rotator. For the affinity purification with antibodies, 2 μg/ml anti-PLCγ2 antibodies were added to the CCL and rotated for at least one hour at 4° C. Following, 25 μl protein A/G-agarose beads (Santa Cruz Biotechnology, 50 % slurry) were added and incubated for one hour at 4° C on a rotator. In the end, beads were washed three times (300 x g, 5 min, 4 °C) with NP40-lysis buffer, mixed with 40 μl 3x Laemmli buffer, incubated for 5 min at 95 °C and subjected to western blot analysis or stored at -20 °C.

3.2.2.7 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

The separation of proteins from CCLs and affinity purification experiments was performed by SDS-PAGE. Therefore, proteins were denatured and reduced using Laemmli buffer (see chapter 3.2.2.1), so that the separation of proteins could be done according to their molecular weight (Weber and Osborn, 1969). Protein samples were concentrated in a 5 % SDS stacking gel (20 mA, 250 V per gel) and separated in a 10 % SDS resolving gel (20 mA, 300 V per gel). The determination of the protein size was done with the help of a prestained protein marker (broad range, 6.5-175 kDa, NEB).

Resolving gel: 8 ml Lower buffer

(1.5 M Tris/HCl pH 8.8, 14 mM SDS, in _ddH₂O) 6 ml 30 % AA/BAA

13.4 ml _ddH₂O 30 μl TEMED 200 μl 10 % APS

41 Stacking gel: 3.75 ml Upper buffer

(0.5 M Tris/HCl pH 6.8, 14 mM SDS, in ddH2O) 2.4 ml 30 % AA/BAA

8.9 ml ddH2O 15 μl TEMED 150 μl 10 % APS

SDS running buffer: 25 mM Tris/HCl 192 mM Glycine 0.1 % (w/v) SDS

in _ddH₂O

3.2.2.8 Western blot analysis

The detection of SDS-PAGE separated proteins was done by western blot analysis.

Therefore, proteins were transferred onto a nitrocellulose membrane by semi-dry blotting (Towbin et al., 1979). To this end, two Whattman paper, a nitrocellulose membrane and the SDS-polyacrylamide gel were soaked in blotting buffer and stacked according to the following arrangement: Whattman paper, membrane, gel, Whattman paper. Importantly, air bubbles were removed by rolling with a glass pipette for efficient blotting. Blotting was performed at 1 mA/cm² for 60 min.

Blotting buffer: 48 mM Tris/HCl

39 mM Glycine

0.0375 % (v/v) SDS

20 % MeOH

in _ddH₂O

3.2.2.9 Immunostaining

Immunostaining was performed to visualize proteins that were previously transferred onto a nitrocellulose membrane. Here, specific antibodies (primary antibodies), which bind to the proteins of interest, and secondary HRPO-coupled antibodies, which recognize the Fc-region of primary antibodies, were used in combination with the ECL detection system for protein visualization. To do this, the membrane was blocked with 5 % BSA in TBS-T for 1 h at RT on a shaker, followed by two washing steps with TBS-T in excess for 5 min each. Incubation with the primary antibodies was done overnight shaking at 4 °C. Next

42 day, the membrane was washed three times with TBS-T in excess and incubated with secondary antibodies for 1 h at RT on a shaker. In the end, the membrane was washed again three times with TBS-T in excess and visualization of proteins was done using 4 ml ECL solution in combination with the Chemilux Camera System (Intas).

TBS-T: 25 mM Tris/HCl pH 8.0 125 mM NaCl

0.1 % Tween20 in _ddH₂O

adjust to pH 7.4

ECL solution: 4 ml Solution A: 250 mg/ml Luminol in 0.1 M Tris/HCl pH 8.6

400 μl Solution B: 1.1 g/L para-hydroxycoumaric acid in DMSO

1.2 μl 30 % H₂O₂