MATERIALS AND METHODS 55
Incubation 60°C 85 min (1 h 25 min)
Denaturation 95°C 5 min
Incubation 60°C 175 min (2 h 55 min)
Hold 20°C Indefinite
After bisulfite conversion was completed, the tubes were briefly centrifuged, and the content transferred into a new 1.5 ml reaction tube. The next steps were performed according to instructions provided with the kit. Finally, bisulfite converted DNA was diluted to 10 ng/µl and aliquots were stored at -80ºC until the Methylight assay was performed. For each PCR reaction 20 ng bisulfite converted DNA was used.
2.6.4 TaqMan-based real time PCR
TaqMan procedure specifically detects the target gene sequence, so nonspecific products do not affect the accuracy of quantification this being the main advantage over DNA binding dyes. TaqMan assays employ a sequence-specific, fluorescently labeled oligonucleotide probe called the TaqMan probe, in addition to the sequence-specific primers.
The probe contains a fluorescent reporter at the 5' end and a quencher at the 3' end. When intact, the fluorescence of the reporter is quenched due to its proximity to the quencher.
During the combined annealing/extension step of the amplification reaction, the probe hybridizes to the target and the dsDNA-specific 5'3' exonuclease activity of nuclease (thermostable polymerases) cleaves off the reporter. As a result, the reporter is separated from the quencher, and the resulting fluorescence signal is proportional to the amount of amplified product in the sample.
Procedure: For the determination of global DNA methylation a TaqMan-based methylation specific real-time PCR (Methylight) system was used. Methylation of LINE1 (Metabion International AG, Germany) and Satα (MWG Operon Eurofins) was determined using the TaqMan Universal PCR Master Mix, No Amp Erase UNG (Roche). As reference for input of bisulfite converted DNA a methylation independent ALU1 (Metabion International AG, Martinsried, Germany) TaqMan system was used. For each real time PCR amplification the template was equivalent to 10 ng of bisulfite converted DNA and measurements were performed in triplicates. Fully methylated bisulfite converted DNA from the EpiTect PCR Control DNA Set (Qiagen GmbH) was used as 100% methylated control. Primers were optimized for StepOnePlus thermocycler. Quantification was normalized to the ALU1 gene within the log-linear phase of the amplification curve obtained for each probe/primer set using the ΔCT method using a reference gene.
MATERIALS AND METHODS 56
Western Blot, also known as immunoblotting or protein blotting, is a method used to detect specific proteins in a complex mixture extracted from cells or tissue. The Western blotting procedure relies upon three key elements (Figure 25): 1. sample preparation and separation of the protein mixture by size using gel electrophoresis; 2. transfer of separated proteins to a membrane; and 3. detection of a target protein by appropriate antibodies. Once detected, the target protein is visualized as a band on a blotting membrane, X-Ray film, or an imaging system.
Figure 25. Schematic overview of the Western Blot method.
2.7.1 Sample preparation from cells
All chemical substances for this assay were purchased from Sigma-Aldrich, if not otherwise mentioned. Protein isolation from cells was made with RIPA (Radio Immuno Precipitation Assay) buffer. The content of the buffer was: 50 mM TRIS-HCl (Merk) (pH=7.5);
150 mM NaCl; 1% TRITON X-100; 0.1% SDS (w/v); 0.5% Natriumdeoxdesoxycholat (w/v) in distilled water. For optimal reproducibility all steps were carried out on ice. For protein isolation from the cells, they were cultivated in 10 cm2 dish (0.5x106 cells). The procedure was as follows: medium was removed, cells were washed once with sterile PBS (PAA Laboratories GmbH), 1 ml of RIPA buffer was added, and incubated 10 min at -20˚C. The cell debris was collected with a cell scraper and the lysate was transferred into a 1.5 ml reaction tube, followed by centrifugation for 20 min at +4°C. After centrifugation the supernatant containing the proteins was collected in a new tube, the protein concentration was measured; aliquots were made and stored at -20˚C until further use.
2.7.2 Sample preparation from tissue
Mr (kDA)
120
10 4 0 110
9 0 5 0 7 0
-
+
120
10 4 0 2 5 9 0 5 0 7 0 110 Mr (kDA)
Target protein
Electrophoresis Transfer to a blotting membrane Detection of the target protein
MATERIALS AND METHODS 57
Histone isolation from fresh carotid tissue was performed with EpiSeeker Histone Extraction kit (Abcam, Cambridge, UK), according to its instructions. Briefly, samples were homogenized with a pre-lysis buffer, and centrifuged at +4°C at 10000 rpm 1 min. The supernatant was removed and the tissue pellet was resuspended in lysis buffer, incubated on ice for 30 min, followed by a centrifugation step by 12000 rpm at +4°C for 5 min. The supernatant was collected in a new tube and supplemented with Balance-DTT buffer. The protein concentration was measured with BCA assay and stored at -20˚C until further use.
Samples were diluted in RIPA buffer to a final concentration of 1 µg/µl. From each sample 20 µl were added into 1.5 ml reaction tubes, together with 5 µl Laemmli buffer 5x (6 ml 1M TRIS-HCl pH=6,8; 10 ml 99% Glycerol; 200 µl 500 mM EDTA; 2 g SDS; 10 mg Bromphenol Blue). Samples were heated for 5 min at 99ºC for denaturation of proteins and incubated on ice.
2.7.3 Protein separation by polyacrylamide gel electrophoresis (PAGE)
The electrophoresis gel is composed of 2 gel types with a different concentration in acrylamide: the stacking gel and the separation gel (Figure 26).
Figure 26. Arrangement of the electrophoresis gel.
Depending on the target protein, different acrylamide concentrations for the separation gel were used as described in Table 14. For the stacking gel the composition is described in Table 15.
Table 14. Separation gel recipes.
Solution
Volume (ml) for 1 gel 15% used for
H3K4me2 and H3K9me2
10% used for OCT4A
6% used for VCAM-1 and VEGFR-2
TRIS-HCl 1.5M(pH=6.8) 2.5 2.5 2.5
Distilled water 2.3 4 5.3
MATERIALS AND METHODS 58
Acrylamide (29:1) 5 3.3 2
SDS10% 0.1 0.1 0.1
Ammonium persulphate (APS)10% 0.1 0.1 0.1
N,N,N′,N′-Tetramethylethylenediamine
(TEMED) 0.005 0.005 0.005
Table 15. Stacking gel recipe.
Solution Volume (ml) for 4ml (1 gel)
TRIS-HCl 0.5M(pH=6.8) 1.25
Distilled water 2.7
Acrylamide (29:1) 0.7
SDS 10% 0.05
APS 10% 0.05
TEMED 0.005
2.7.4
Protein transfer to PVDF membrane
After the electrophoresis was finished, the procedure was continued by protein transfer onto a PVDF membrane using semi-dry system. The scheme of this procedure is shown in Figure 27.
Figure 27. Assembling of the transfer sandwich.
An aqueous solution of the transfer buffer was prepared by adding following substances/solutions to 900 ml distilled water (B. Braun Melsungen AG): 3.03 g Trizma base, 14.4 g glycine, and 100 ml methanol (Merck). The filter papers and acrylamide gel were soaked in transfer buffer; the PVDF membrane was shortly activated in methanol and incubated in transfer buffer for 30 min. The assembling of the system was made as described in Figure 27. The semi-dry transfer device was set to 10V for 30 min followed by other 5 min at 15V.
2.7.5 Protein detection
MATERIALS AND METHODS 59
The PVDF membrane containing the proteins was then incubated in blocking buffer (5% non-fat skimmed milk powder (Biomol GmbH, Hamburg, Germany) in TBS-T) for 1 h at RT on a shaker. TBS-T was prepared from 900 ml distilled water to which following solution were added: 100 ml TBS buffer, and 1 ml Tween 20. TBS buffer was prepared by adding 24.2 g Trizma base and 80 g NaCl (Merck) to 1000 ml distilled water and adjusting the pH value to 7.6 with HCl 2N (Apotheke MRI). Each antibody was diluted in a blocking buffer and incubated over night at +4˚C as described in Table 16, 17 and 18.
The next day the membrane was washed in TBS-T solution for 30 min to remove the excess of the first antibody. The secondary antibody solution was made in the same blocking buffer and added to the PVDF membrane, which was then incubated for 1 h at RT.
Table 16. Antibodies used for characterization of the angiogenesis in adMSCs.
Name Reacts with Clone/Made
in Brand &Cat.
Nr. Dilution Size (kDa) Mouse anti hVACM-1(CD106) Human Mouse IgG R&D Systems 1:100 110
Flk-1 antibody(VEGFR-2) Human Mouse
IgG1 R&D Systems 1:100 170 Anti-GAPDH antibody [6C5] Rat, Rabbit,
Chicken, Human,
6C5 Abcam
(ab8245) 1:1000 36 Peroxidase-Labeled Affinity
Purified Antibody to Mouse IgG
(H+L)(Human Adsorbed) Mouse Goat KLP
(074-1806) 1:5000 - Peroxidase-Labeled Affinity
Purified Antibody to Rabbit IgG
(H+L)(Human Adsorbed) Rabbit Goat KLP
(074-1516) 1:5000 -
Table 17. Antibodies used for detection of pluripotency markers.
Name Reacts with Clone/Made
in Brand &Cat.
Nr. Dilution Size (kDa) OCT4A(C30A3) Rabbit mAb Human, Mouse C30A3 Cell Signaling
(#2840) 1:500 48 Rabbit polyclonal to Nanog Human, Mouse rabbit Abcam
(ab21624) 1:500 -
1:50 38
Anti-GAPDH antibody [6C5] Rat, Rabbit, Chicken,
Human,
6C5 Abcam
(ab8245) 1:1000 36 Peroxidase-Labeled Affinity
Purified Antibody to Rabbit IgG
(H+L)(Human Adsorbed) Rabbit Goat KLP
(074-1516) 1:5000 -
Table 18. Antibodies used for detection of di-methylation at K4 and K9 sites of histone H3.
Name Reacts with Clone/Made
in
Brand &
Cat. Nr. Dilution Size(kDa)
MATERIALS AND METHODS 60
Di-Methyl-Histone H3
(Lys4)(C64G9) Rabbit mAb Human, Mouse ,
Rat, Monkey Rabbit IgG
Cell Signaling
(#9725) 1:3000 17 Di-Methyl Histone H3(Lys9)
Antibody
Human, Mouse,
Rat, Monkey Rabbit IgG
Cell Signaling
(#9753) 1:200 17 Peroxidase-Labeled Affinity
Purified Antibody to Rabbit IgG (H+L)(Human Adsorbed)
Rabbit Goat KLP
(074-1516)
1:5000 -
The PVDF membrane was treated for 30 min with TBS-T then submersed in a working solution (Solution A/Solution B, ratio 1/1) of SuperSignal® West Pico Chemiluminescent Substrate (Thermo Scientific, Bonn, Germany) for 5 min. The excess reagent was drained and the blot was covered with clear plastic wrap. The membrane was placed in a film cassette with the protein side facing up. In the dark, a FUJI Medical X-Ray Film (FUJIFILM Europe GmbH, Düsseldorf, Germany) was placed on top of the membrane and various exposure times were used to achieve optimal results. The film was then developed with Medical Film Processor (Konica Minolta model, SRX-101A, Konica Minolta Medical and Graphic Imaging Europe GmbH, Munich, Germany)
2.7.6 Protein quantification
In order to quantify the amount of each targeted protein, developed films were digitized using a color image scanner (HP Scanjet 3800). The bands on the films with the same exposure time were compared to each other after a previous normalization to a housekeeping gene for GAPDH. The intensity quantification was made with ImageJ software (National Institutes of Health, Bethesda, USA).