Since proteins are highly temperature-sensitive, all protein work including thawing was performed on ice. Since proteins can furthermore degrade through protease activity, protease inhibitor was added to the RIPA buffer.
4.12.1.1 Isolation of proteins from mouse tissues
For protein isolation from mouse tissue, a small part of the respective tissue was lysed in 350-600 µl RIPA buffer, depending on the size of the sample. Prior use, complete mini protease inhibitor (1 pill per 7 ml of RIPA buffer) was added to the RIPA buffer to block protease activity. Soft tissues like spinal cord or brain were homogenized with help of an EPPI-pestle. For compact tissues like muscle, the T 10 basic ULTRA-TURRAX homogenizer was used. Next, protein solutions were centrifuged for 30 min at 13.200 rpm at 4°C. The supernatant containing the dissolved proteins was transferred to a fresh tube and the protein concentration was determined (chapter 4.12.1.3).
4.12.1.2 Isolation of proteins from cells
To harvest proteins, cells were first washed two times in 1 x PBS. Lysis of the cells was performed by adding 50 µl RIPA buffer containing protease inhibitor (chapter 4.12.1.1) directly on a 10 cm Petri dish (30 µl per 1 well of a 6 well plate). The dishes were kept on ice for 20 min before a cell scraper was used to collect the solution which was then transferred into a fresh 1.5 ml tube. Following another 20 min of incubation on ice, the tubes were centrifuged for 20 min at 13.200 rpm at 4°C. The supernatant was transferred into a new 1.5 ml tube and the concentration was determined (chapter 4.12.1.3) before it was frozen at -80°C.
4.12.1.3 Determination of protein concentration
The protein concentration was determined using the Bradford method (Bradford, 1976).
Bradford dye is able to bind to proteins whereupon it shifts its absorption maximum from 470 to 595 nm. To determine the protein concentration of a sample, 2 µl of protein lysate were mixed in 498 µl of Bradford solution and incubated for 20 min at RT. After blank with Bradford dye, the absorption of the sample was measured at a wavelength of 595 nm. The protein concentration of the unknown sample was determined by the mashine comparing the measurements to a bovine serum albumin (BSA) standard curve.
4.12.1.4 SDS polyacrylamide gel electrophoresis (SDS PAGE)
When separated by SDS PAGE, proteins need to be denatured in Laemmli buffer and under the influence of heat (95°C). The SDS of the Laemmli buffer and the PAA gel applies a
negative charge to the protein which by far exceeds the intrinsic charge of the protein. This way, SDS page allows the separation of all proteins of a sample by size (Laemmli, 1970).
For SDS PAGE, a 12 % PAA separation gel (chapter 4.7.6) was prepared between two glass plates (0.5 mm thickness). Isopropanole was pipetted on top to cover the gel. After polymerization, the isopropanole was removed and a stacking gel (chapter 4.7.6) was poured on top of the separation gel. A suitable comb was fixed between the glass slides and adjusted using two clamps. Stacking- and separation gel differ by their PAA content and pH.
Since the stacking gel contains less PAA it forms bigger pores through which the denatured proteins can migrate easily in between a front of dipolar glycine and Cl- ions. Therefore, the function of a stacking gel is to concentrate the proteins along the border of stacking and separation gel. Once the proteins migrate into the separation gel, the glycine dissociates due to the increased pH. The separation gel contains a bigger amount of PAA and therefore smaller pores are formed. Thus, bigger proteins are slowed while small proteins can easily migrate through the pores. Due to their overall negative net charge, proteins are separated only by size. Protein samples to be separated (typically 7.5 µg) were supplied with 5 µl of 2x Laemmli buffer to which β-Mercaptoethanol had been added in a ratio of 1:10. After mixing and a short spin to collect the solution at the bottom of the tube, the proteins were heated to 95°C for 5 min for complete denaturation. The heating step was followed by a short centrifugation step at 4°C to collect condensed fluid from the tube seam. Proteins were kept on 4°C until loaded on the gel. Protein size was estimated loading the PAGE Ruler Plus (Fermentas) protein ladder in a separate lane. Gel electrophoresis was performed using the Mini-Protean 3 cell or Protean II xi system (Biorad). The proteins were run at 40 V in 1 x electrophoresis buffer (4.7.6) until reaching the separation gel. From this point on, the applied current was increased to 80-120 V.
4.12.1.5 Transfer of proteins to a nitrocellulose membrane (Western blotting)
To transfer proteins on a nitrocellulose membrane by wet blotting, the separation gel was removed from the glass plates and the remaining stacking gel was discarded. Two sponge pads, two filters, the nitrocellulose membrane as well as the gel were equilibrated in transfer buffer (4.7.6) and arranged on top of each other in the following order: Sponge pad, filter, gel, membrane, filter, sponge pad. Possible air bubbles in the sandwich were removed by carefully rolling a small glass pipette over the filter paper. The gel sandwich was then inserted into a transfer cell and a cooling module was added. The transfer cell was placed in a 4°C room and a current of 30 V was applied o.n..Caused by the negative charge, the proteins migrate in the electric field and finally adhere to the membrane due to hydrophobic interactions, at the same time keeping the separation pattern present in the gel. The next morning, the wet blot was disassembled and the membrane was briefly rinsed in TBST.
Protein transfer was either monitored via reversible Ponceau staining or the membrane was immediately used for subsequent immunological detection.
4.12.1.6 Ponceau staining of proteins on nitrocellulose membranes
To control the protein transfer to the nitrocellulose membrane staining with Ponceau solution is commonly used. Ponceau interacts with positively charged amino acid side chains and since the staining is reversible the membrane can afterwards be used for subsequent immunological detection using antibodies. For Ponceau staining, the membrane was incubated in Ponceau solution for 30 sec. The arising band pattern could be clarified rinsing the membrane in fresh TBST. For further analysis, the membrane was simply washed in TBST until no red signal was present on the membrane any longer.
4.12.1.7 Immunological detection of proteins on nitrocellulose membranes
To detect proteins on a nitrocellulose membrane, primary antibodies are used that specifically recognize the protein of interest. The staining procedure was performed as follows: After wet blotting, the membrane was washed 5 times in TBST to remove any residual MeOH from the transfer buffer. To avoid background, the membrane was next blocked using 6 % non-fat milk powder in TBST for 3 h. The 6 % non-fat milk was discarded and exchanged with 3 % non-fat milk in which the primary antibody had been diluted. After incubation with the primary antibody, the membrane was washed 5 x for 5 min in TBST to remove unbound primary antibody. Following washing, the membrane was incubated with an HRP-conjugated secondary antibody in 3 % non-fat milk that was directed against the species from which the primary antibody was derived. After incubation with the secondary antibody, the membrane was washed again 5 x for 5 min in TBST to remove unspecifically or unbound secondary antibody. To chemiluminescently detect the protein bands, the membrane was incubated for another 5 min in SuperSignal® West Pico Chemiluminescent Substrate (Pierce). Lastly, the membrane was carefully wrapped in plastic foil, placed into an developing cassette and exposed to Hyperfilm ECL (Amersham). Densitometric analysis of the films was performed using ChemiDOC XRS analysis software.
For a summary of primary and secondary antibodies used in this study and respective dilutions and incubation times see Table 12. For order numbers of the respective antibodies refer to chapter 4.6.
Table 12: Antibodies used in Western blotting
Primary antibodies Dilution Incubation time
Rabbit-α-PLS3
(custom made, Eurogentech) 1: 4,000 o.n.
α-SMN (monoclonal) 1:2,000 2 h, alt. o.n.
α-V5 (monoclonal) 1:3,500 o.n.
α-V5-HRP conjugated 1:1,500 2 h
α-Actin (monoclonal) 1:80,000 0.5 h
α-GAPDH (monoclonal) 1:4,000 o.n.
Rabbit-α-β-Tubulin (polyclonal) 1:80,000 0.5 h
Secondary antibodies Dilution incubation time
Goat-α-mouse-HRP 1:10,000 1 h
Donkey-α-rabbit-HRP 1:10,000 1 h