Biochemical analysis of protein extracts:

2.5.1. Protein concentration determination by Bradford Assay:

Bradford Assay was used to determine the total protein concentrations in the tissue and in the cell lysates. 200μl of 5x Bradford reagent (Bio-Rad, Germany) was mixed with 800μl of water in an eppendorf tube, and 4μl of protein sample was added to the mixture. The reaction mixture was incubated for 5 minutes, transferred to UV spectroscopy cuvette and absorbance was measured at 595 nm. Bradford reagent (1x) without protein sample served as blank. The protein concentration of the samples was calculated from the BSA standard reference curve generated by measuring the known concentrations of BSA (Bovine serum albumin). BSA standard curve was made by measuring BSA (1mg/ml) in the range of 2-16 μg at 595 nm.

2.5.2. Semi-quantitative western blot analysis:

2.5.2.1. SDS Poly-Acrylamide Gel Electrophoresis:

Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the standard protocol established by (Laemmli, 1970). SDS-PAGE is a commonly used biochemical method to separate proteins according to their molecular mass: by the differential rate of migration of proteins through the matrix (polyacrylamide gel). This method is based on the principle that a charged molecule moves towards its opposite charge electrode under a constant electric field. Sodium Dodecyl Sulfate (SDS) is an amphipathic surfactant molecule with an anionic head group and a 12carbon tail attached to the anionic head group. SDS denatures and linearizes the proteins by binding to the amino acid chain with its hydrocarbon tail. The SDS imparts an overall negative charge to the proteins so that

structure and linearize, the proteins (see section 2.2.2 for the composition of buffers and solutions used.)

The matrix for the separation of proteins was made of polyacrylamide (acrylamide/bisacrylamide) as it is inert in nature and could be easily made at different concentrations for variable pore size. The polymerization of acrylamide/bisacrylamide takes place by free radical mechanism initiated by Ammonium peroxodisulfate (APS) in the presence of N,N,N′,N′- Tetramethylethylene-1,2-diamine (TEMED). Tris-HCl buffer was used for pH adjustment. The polyacrylamide gel was prepared in between two glass plates. The dimensions of the gel were 55 mm x 85 mm. The gel was divided into two parts, upper "stacking gel" and the lower one "running gel". The stacking gel has a low concentration of acrylamide/bisacrylamide and low pH of 6.8. The running gel has pH of 8.8 and acrylamide concentration ranging from 5% to 12.5% depending upon the size of the proteins to be separated (10% is optimum for proteins ranging from 100kDa to 25kDa). The running gel is prepared first and poured between the glass plates; covered with ethanol to avoid the contact with oxygen for the polymerization of the gel. Polymerization takes approximately 15-20 minutes. After the running gel has polymerized completely, ethanol was removed and stacking gel was poured and the comb was inserted. The polyacrylamide gel was run in glycine buffer with pH 8.3.

Protein samples (40 μl) were mixed with 8μl of with 6x reducing buffer (see section 2.1.2) and fresh DTT (50 mM) and boiled at 95°C for 5 minutes. After the protein samples reached room temperature, it was loaded in the gel pockets along with the standard protein ladder of known molecular weight for reference. A constant current of 15 mA was applied until the protein sample reached running gel part;

subsequently, the current was increased to 30 mA and the gel was allowed to run till the samples reach the end of the running gel (around 50-60 minutes).

2.5.2.2. Semi-dry western blot:

The proteins were separated by SDS-PAGE as described in section 2.5.2. The separated proteins were transferred from the gel to a support membrane made of nitrocellulose (Amersham Protran 0.45 micron, GE Healthcare, Germany) for further analysis by Immunostaining. A transfer sandwich was prepared as shown in Fig. 2.1 to transfer the proteins from the gel to the membrane. Transfer sandwich consisted of three filter papers (Filter paper 330 g/m², Sartorius Stedim Biotech GmbH, Göttingen), nitrocellulose membrane and polyacrylamide gel without stacking part, soaked in anode buffer (see section 2.1.3) and three filter papers soaked in cathode buffer (see section 2.1.3). The transfer of the proteins was done at a constant electric field of 1mA/mm² (dimensions of the gel 55 mm x 85 mm) for 30 minutes to 120 minutes depending upon the size of the proteins to be transferred. The proteins with molecular mass of 20-25 kDa required 30 minutes of transfer time while 200-250 kDa proteins required 120 minutes.

Figure 1.2 Semi-dry western blot transfer sandwich: The transfer sandwich was assembled as shown. The flow of proteins takes from cathode to anode as the proteins extracted are negatively charged imparted by SDS. (Figure adapted from GE healthcare website)

2.5.2.3. Immunostaining of proteins bound to the nitrocellulose membrane

The proteins in the tissue/cell extracts were separated and transferred to the nitrocellulose membrane as described in section 2.5.2.1 and 2.5.2.2, respectively.

The proteins on the membrane were detected by specific primary antibody followed by an appropriate secondary antibody. The membrane was incubated in blocking solution (5% skimmed milk in TBST) for 1 hour at room temperature with gentle shaking. Primary antibody was diluted to the desired concentration in blocking solution and the membrane was incubated overnight at 4ºC with gentle shaking.

Primary antibody was removed and can be reused for up to 6-8 times; the membrane was washed with blocking solution, three times 10-15 minutes each and incubated with appropriate HRP (Horse Radish Peroxide) coupled secondary antibody in blocking solution (1:10000) for 1 hour at room temperature with gentle shaking.

The secondary antibody was removed; the membrane was washed with TBST (see section 2.1.3), three times for 10-15 minutes. The membrane was incubated in chemiluminescent substrate solution (see section 2.1.3) for 5 minutes and imaged by Intelligent Dark Box II camera (Fuji Film, Japan)

2.5.2.4. Stripping of nitrocellulose membrane

The proteins on the nitrocellulose membrane were detected by immunostaining as described in section 2.5.2.3. The membrane was reused for probing with different antibodies by removing the primary and secondary antibody under the mild condition to minimize any loss of proteins. The membrane was incubated in Glycine stripping buffer, pH 2 (glycine, SDS, tween 20, see section 2.2.3.7) for 10 minutes at room temperature with gentle shaking; washed with PBS (see section 2.1.3) twice for 10 minutes followed by washing with TBST twice for 10 minutes.

The membrane was then ready for Immunostaining as described in section 2.5.2.3.

2.5.2.5 Primary antibodies

Antibody Epitope Species Source Dilution

Amphiphysin aa 2-15 Rabbit Synaptic System 1:1000

Arf6 peptide aa 150

C-ter

Rabbit Abcam 77581 1:1000

Endophilin A1 mouse aa 256-276 Rabbit SYSY 159 002 1:1000

Git 1 Rat aa 375-770

Hsp110 Haster aa703-858 Mouse BD 610510 1:1000

Hsp90 α Human peptide Rabbit Bioss bs-10100R 1:100 Intersectin-1 mouse aa 800-909 Mouse BD 611574 1:500

LRRK2 Human aa

γ1-adaptin BD C-term/ear-domain Mouse BD 610386 1:2000