3. METHODS
3.3. Protein Techniques
Methods
ligation the isolated RNAs have to be phosphorylated at their 5’end. Therefore, reagents listed in table 3-28 were added to 10 µl RNA and the mixture was incubated at 37 °C for 30 min.
Table 3-28 Phosphorylation Reaction Mixture for Small RNA Cloning
Reagents Amount
10x PNK buffer 2 µl ATP [100 mM] 0.4 µl
PNK 0.5 µl
DEPC H2O 7.1 µl
Following this, 37 µl DEPC-H2O and 3 µl 5 M NaCl solution were added and RNA was extracted with PCI (see 3.1.9). Ligation of the 5’-linker was done in the same way as the ligation of the 3’-linker with subsequent purification on a 8.5 cm 15% polyacrylamide gel containing 8 M Urea. RNA in the range of 85-91 nt was cut out and isolated as described above. The RNA was then reverse transcribed to cDNA using the 3’-outer primer (see also 3.1.11.3). For unspecific amplification of the ligated small RNAs a PCR was carried out with the 5’- and 3’-outer primers (3.1.10). If the yield was too low a second PCR was performed by using the 5’- and 3’-inner primers. To sequene the 5’-ends of single cloned small RNAs, products were PCR amplified with a specific 3’-primer for each identified miRNA and the 5’-outer or -inner primer. Products were purified on 2% agarose gels, extracted, cloned into a TA-cloning vector (3.1.6.1) and then sequenced (3.1.8).
3.3.3. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
For western blotting 10-50 μg protein were loaded per lane of an SDS-gel. To separate proteins electrophoretically, they were dissolved in Laemmli buffer containing negatively charged SDS, which compensates the positive charge on denatured proteins. Within a SDS-gel the proteins move towards the anode in an electric field. The velocity of a protein through the pores of a gel is determined by its size. To increase the separation of proteins, a lower concentrated gel (collecting gel) is polymerized above a higher concentrated gel (separating gel) to concentrate proteins at the boundary before separation (Laemmli, 1970). The gel Components are shown in table 3-30 and 3-31.
Table 3-29 Collecting Gel Buffer (10x)
Reagents Concentration
TRIS base 0.47 M
H3PO4 to pH 6.7
Table 3-30 Separating Gel Buffer (10x)
Reagents Concentration
TRIS base 3 M
HCl to pH 8.9
Table 3-31 SDS-Polyacrylamide Collecting Gel
Reagents Amount
Acrylamide Stock (Roti30) 1 ml Collecting gel buffer (10x) 1.25 ml
SDS [10%] 100 μl
APS 100 μl
TEMED 25 μl
H2O to 10 ml
Table 3-32 SDS-Polyacrylamide Separating Gel [14%]
Reagents Amount
Acrylamide Stock 9.3 ml Separating gel buffer (10x) 2.5 ml
SDS [10%] 200 μl
APS 70 μl
TEMED 12.5 μl
H2O to 20 ml
Table 3-33 TGS Buffer (Tris Glycine SDS) (1x)
Reagents Concentration
TRIS base 25 mM
Glycine 192 mM
SDS 0.1%
pH 8.3
Methods
Table 3-34 Laemmli Sample Buffer (5x)
Reagents Concentration
SDS 10% (w/v)
Glycerol 50%
DTT 10% (w/v)
Bromo phenol blue 0.05% (w/v)
Samples were first resuspended in an appropriate volume of H2O (e.g. 10 µl for 1-2x 106 cells) and mixed with an equal amount of 5x Laemmli buffer. The mixture was heated to 95 °C for 5 min and stored on ice before loading. Samples were loaded onto the gel and run at 80 V for 20 min in 1x TGS.
After the dye front reached the boundary to the separating gel the voltage was increased to 160 V for another 40-60 min until the blue front reached the end of the gel. Proteins were then blotted onto a nitrocellulose membrane.
3.3.4. Western Blot
The separated proteins on the SDS gel were transferred to a nitrocellulose membrane. The positively charged membrane binds and immobilizes negatively charged proteins. The membranes can then be probed for the detection of different proteins with specific antibodies.
The transfer was performed using a semi-dry set up. Whatman papers (3 mm) and a cellulose membrane were cut to the same size as the gel. The Whatman paper as well as the membrane and the gel were immersed in 1x TGS buffer with 20% Ethanol. In a Phase Transfer System the Components were stacked as follows: three Whatman papers, then the gel, the membrane and 4 Whatman papers.
Air bubbles were carefully squeezed out. The transfer was performed with a current of 0.8 mA/ cm2 for 2 h.
Complete transfer of proteins was analyzed using Ponceau S solution, to stain proteins on the membrane. Ponceau stain is reversible and was performed for 5 min under agitation. Afterwards the membrane was washed for 10 min in 1x TBS (Tris Buffered Saline) before continuing.
To minimize unspecific binding of the antibodies to the membrane, the membrane was first blocked by incubation in 10% milk powder in TBS (MTBS) for 30 min under agitation. Afterwards the 1st antibody was diluted in 5% milk powder in TBST (TBS, Tween) (MTBST) and the membrane was incubated for 2 h at RT or over night at 4°C under constant agitation. Membranes were then washed 5 times for 5 min in 1x TBST and pre blocked in 10% MTBS for 15 min before incubating with the 2nd antibody in 5% MTBST for 2 h at RT. Subsequently membranes were washed again 5 times in 1x TBST for 5 min, twice in 1x TBS and once in H2O.
Since the secondary antibodies are coupled to horseradish peroxidase the detection of protein bands was accomplished by adding chemiluminescent substrates and measuring the resulting signal.
Therefore ECL solution A and B were mixed in equal amounts, membranes were incubated in the solution for 2 min and the chemiluminescent signal was detected on anX-ray film.
Table 3-35 Blotting Buffer (1x)
Reagents Concentration
TRIS base 25 mM
Glycine 192 mM
SDS 0.01% (v/v)
Ethanol 20%
to pH 8.6
Table 3-36 TBS Buffer (1x)
Reagents Concentration
TRIS base 100 mM
NaCl 0.9% (w/v)
HCl to pH 7.5
Table 3-37 TBST Buffer (1x)
Reagents Concentration
TRIS base 100 mM
NaCl 0.9% (v/v)
HCl to pH 7.5
Tween 20 0.1% (v/v)
Table 3-38 ECL Solution A
Reagents Concentration
TRIS-HCl, pH 8.5 100 mM Cumaric acid 0.4 mM
Luminol 2.5 mM
Table 3-39 ECL Solution B
Reagents Concentration
TRIS-HCl, pH8.5 100 mM
H2O2 0.18%
3.3.5. Immunoprecipitation RIP-ChIP
The RIP-ChiP was performed after a protocol previously described (Keene et al., 2006). Cells (1-2x 107) were infected with adenovirus (Ad-IRES or Ad-BART) at an MOI of 500. After 2 days, the cells were collected by scraping in ice cold PBS and pelleted by centrifugation (1000 g, 3 min, 4°C). Cells were washed three times with ice cold PBS and pelleted as described above. Cells were then lysed in one pellet volume of polysome lysis buffer (table 3-40) supplemented with RNAse inhibitors (RNAseOut and Vanadyl ribonucleoside complexes (VRC)) as well as protease inhibitors. The mRNP lysate was incubated on ice for 5 min and immediately frozen at -80 °C for at least 30 min or until further needed. Magnetic beads (50 µl per IP reaction) were washed with 200 µl PBS, pelleted and blocked in 200 µl NT2 buffer (table 3-41) supplemented with 5% BSA for 10 min. The beads were then pelleted and the blocking step was repeated. Next 200 µl NT2 buffer containing 5% BSA and
Methods
The next day the lysate was thawed on ice and centrifuged (15000 g, 15 min, 4 °C). The supernatant was transferred into a new tube and stored on ice until needed. The lysate was pre-cleared with 25 µl beads, which were washed and blocked in a similar fashion as the beads for anitbody incubation, resuspended in 850 µl ice cold NT2 buffer containing 200 U RNaseOut, 400 µM VRC, 1 mM DTT and 20 mM EDTA and incubated for 30 min at 4 °C under rotation.
The antibody coated beads were gently washed 5 times with ice-cold NT2 buffer. The beads were then resuspended in 950 µl of pre-cleared lysate and 100 µl was saved as an input control. The beads were incubated for 4 h at 4 °C under rotation. The beads were then pelleted, the supernatant was removed and they were washed 5 times with ice cold NT2 buffer as described above. Following this, the beads were resuspended in 100 µl NT2 buffer containing 30 µg Proteinase K and incubated at 55 °C for 30 min. The tubes were occasionally agitated. Then 1 ml of RNA Bee was directly added and RNA isolation was performed as described in chapter 3.2.1. For precipitation, 20 µg of glycogen were added in the precipitation step. RNA was solubilized in 5-10 µl DEPC-H2O.
Table 3-40 Polysome Lysis Buffer
Reagents Amount [for 5 ml]
KCl [1 M] 500 µl
MgCl2 [1 M] 25 µl HEPES [1 M] 50 µl
NP40 25 µl
DTT [0.1 M] 50 µl RNAseOut [40 U/µl] 12.5 µl Vanadyl ribonucleoside complexes [200 mM] 10 µl Protease inhibitors 200 µl
Table 3-41 NT2 Buffer
Reagents Amount [for 50 ml]
TRIS-HCl [1 M], pH 7.4 2.5 ml
NaCl [1 M] 7.5 ml
MgCl2 [1 M] 50 µl
NP40 25 µl