Western blot analysis

2.3.3.1 Protein extraction

All steps of protein extraction were performed at 4°C. For cell lysis, growth medium was removed and the cells were washed twice with 5 ml cold PBS to remove all residual medium.

Gen Primer sequences

Forward primer Reverse primer

CXCL12 5´- GGT CGT GGTCGTGCTGGT -3´ 5´-CGG GCT ACA ATC TGA AGG G -3´

CXCR4 5´-TAC ACC GAG GAA ATG GGC TCA -3´ 5´- AGA TGA TGG AGT AGA TGG TGG G-3´

HPRT1 5´-TGA CAC TGG CAA AAC AAT GCA -3´ 5´-GGT CCT TTT CAC CAG CAA GCT -3´

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The cells were then scraped off the flask bottom with a cell scraper and resuspended in PBS.

This suspension was centrifuged at 1200 rpm for eight minutes at 4°C. After the centrifugation, the PBS supernatant was removed.

500 μl of lysis buffer (see the composition of the lysis buffer in Table 2.10) was added to the cell sediment and stirred carefully. Ultrasound was used to facilitate cell lysis and protein release with the following parameters: four times with duration of 0.9 seconds and 42 % intensity.

Table 2.10: Composition of the lysis buffer

Substrate Molecular weight (MW) Dilutions

20 mM Tris HCl (pH=7.5) 157.60 0.0315 g/100 ml water

150 mM NaCl 58.60 0.0876 g/100 ml water

1 mM MgCl2 203.30 0.002 g/100 ml water

1 mM CaCl2 147.02 0.0014 g/100 ml water

1% NP-40 - 1 ml/100 ml water

10% glycerol - 10 ml/100 ml water

10 ml lyse buffer

+ 1 pill of Mini, EDTA-free, protein inhibitor

The cell-buffer suspension was incubated on ice for ten minutes in order to let the foam subside. A series of five to six passages through a fine insulin cannula was done manually to increase protein release. This step was repeated after ten minutes. After the final passage, the suspension from each tube was transferred into a nonsterile, 1.5 ml plastic cup and centrifuged at 6000 rpm for ten minutes at 4 °C.

The lysate was transferred to a fresh 1.5 ml cup for Bradford assay and western blot. Lysates were kept on ice until use or at -80°C for long-term storage.

2.3.3.2 Bradford assay

Protein concentrations in the samples, were determined by Bradford assay (Bradford 1976).

Bovine serum albumin (BSA) was used to prepare a standard curve (with 0 to 2 mg/ml protein

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concentration range). First a standard curve was prepared, by defining the standard absorbance values on the y-axis and their concentrations in mg/ml on the x-axis, in order to determine unknown protein concentrations. One millilitre Bradford solution (Bio-Rad) was added to each vial and mixed by inversion. The blank sample was 20 μl sterile water in one millilitre of Bradford solution. The absorbance of the prepared standard concentration samples were measured at 595 nm and the standard curve was drawn. For samples with unknown protein concentration, 20 μl of the samples was added to one millilitre Bradford solution and the absorbance was measured. The values were entered into the standard curve to obtain the protein concentration of the sample.

The measurements were performed in duplicate to improve the accuracy of the determination.

The results corresponded to the protein concentration in 20 µl of lysate. The amount of lysate required for loading the electrophoresis gel was calculated by dividing 20 µl of lysate volume by the concentration. In order to reach a final volume of 20 µl, we calculated the volume of sodium dodecyl sulphate (SDS) buffer that had to be added (see the SDS buffer consistence in Table 2.11).

2.3.3.3 Gel electrophoresis

Purified proteins were separated on precast 10-well polyacrylamide gels (TGX-gel). The gels were placed in the migration set that consisted of a tank, lid, and an electrode assembly. The separated proteins were further processed for western blotting using a Trans-Blot Turbo^TM system.

Adequate volumes of the protein lysate and SDS buffer-mercaptoethanol solution (see Table 2.11) were mixed together. These mixtures were centrifuged for a few seconds by rapid acceleration. Afterwards they were incubated for five minutes at 95 °C.

Samples were loaded into wells of the TGX-gel and run against protein standards. The gel electrophoresis was performed at 200 V and 30 mA. Once migration was over, the proteins were transferred from the gel onto a nitrocellulose membrane.

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Table 2.11: SDS buffer consistence

2.3.3.4 Western blotting

After the proteins had been separated by electrophoresis, they were transferred by electroblotting to a nitrocellulose membrane. In electroblotting an electric current is used to propel the proteins from the gel onto the nitrocellulose membrane while maintaining their pattern.

Electroblotting was done with the Trans-Blot Turbo^TM Transfer system following the manufacture’s instruction using a transfer time of three minutes. The protein-loaded membranes were then stained with the sodium salt of a diazo dye. The loading and transfer efficiency was analysed with this procedure. The dye was later washed out with water.

Western blotting of the nitrocellulose membrane was performed after protein transfer. Prior to incubating the protein-loaded membranes with the primary antibody, the membrane-blocking step is crucial to avoid non-specific primary antibody binding in protein in free spaces. For this purpose a WesternBreez^® Chromogenic Western blot immunodetection kit was used. The membrane was blocked with blocking solution for 30 minutes on a rotatory shaker followed by two washes of five minutes with distilled water.

Following this, the primary antibody was incubated on the membrane for one hour at room temperature or over-night at 4°C. For the present study, rabbit anti-CXCR4 monoclonal antibody (see Table 2.5) was used.

Beta-actin, as a ubiquitous structural protein, was used to confirm equal loading of the protein lysates. Due to an almost identical molecular weight of the two targeted proteins (molecular weights of CXCR4 and β-actin proteins were approximately 40 and 42 kDa, respectively), western blotting was performed to detect each protein separately on two individual

SDS buffer (pH= 7.4) 30 mM Tris-Base 9 % SDS

15 % glycerine

0.04 % bromphenol blue Na-salt 10 % 2-mercaptoethanol

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membranes. For this purpose monoclonal mouse anti-β-actin antibody was applied as primary antibody (Table 2.5). The primary antibodies were diluted in five millilitres of blocking solution in order to obtain the dilutions recommended by the manufacturers. For anti-CXCR4 a dilution of 1:500 and for anti-β-actin a dilution of 1:10 000 was used.

An incubation time of one hour was kept. After the membranes had been washed three times for five minutes each in ten millilitres of antibody wash to remove residual primary antibody, the membranes were re-incubated for 30 minutes in five millilitres of the secondary antibody solution. The choice of the secondary antibody depended on the primary antibody (i.e. mouse or rabbit). In order to remove residual secondary antibody the membranes were washed three times for five minutes followed by a final wash procedure of three two-minute washes with distilled water.

In the last step, the membranes were incubated with 5 ml of chromogenic substrate for one to 60 minutes without agitation. With our antibodies, 15 minutes were enough to visualise the bands.

The membranes were then washed for a final three times in ten millilitres of distilled water for two minutes before placing them on a clean filter paper to dry in the open air at room temperature.

The stained bands were visually compared to those of the marker loaded during electrophoresis to estimate size. The process was performed with the above mentioned β-actin protein-loaded membrane. Beta-actin was therefore also useful in detecting eventual errors such as incomplete transfers.