Laemmli buffer

For western blot analysis, Drosophila heads were homogenized in 15 μl of 2x Laemmli buffer (Sigma-Aldrich). Samples were boiled for ten minutes and subjected to SDS-PAGE. Protein were transferred onto nitrocellulose membranes (Bio-Rad), blocked in 2% milk in PBS with 0.05% Tween 20 (PBSTw), and incubated overnight at 4°C with primary antibodies. Membranes were washed three times with PBSTw and incubated for three hours with appropriate HRP-conjugated secondary antibody (SouthernBiotech) at room temperature. Following multiple washes with PBSTw, proteins were visualized by enhanced chemiluminescence (Alpha Innotech). Protein transfer and loading were monitored by Ponceau S staining and by reprobing blots with an antibody directed to GAPDH. All immunoblots were repeated at least three times. Primary antibodies to the following proteins were used at the indicated concentrations: α-synuclein (1:6,000,000, H3C, Developmental Studies Hybridoma Bank), GAPDH (1:40,000, Invitrogen), cofilin (1:1,000,000, Anna Marie Sokac).

For each test sample, 50 hermaphrodites at day 1 and 4 of adulthood were transferred into an Eppendorf tube filled with 1 ml M9 buffer. Worms were spun at 800 rpm for 2 min and supernatant removed, leaving 25 μl. Then 25 μl of 2x Laemmli buffer (Sigma) was added, and samples incubated at 70°C for 15 min, vortexed every other minute and then shifted to 95°C for 5 min. Lysates were centrifuged at 13,000 rpm at 4°C for 15 min. 20 μl of each sample was loaded onto a Criterion XT Tris-Acetate gel. The gel was run at 200V in SDS-PAGE chamber with 1x 3-(N-morpholino) propanesulfonic acid (MOPS) buffer for 45 min. The gel was fixed in methanol, acetic acid and ultrapure water in ratio of 50:10:40 for 30 min. The fixing solution was then discarded and replaced with Coomassie fixation solution (50:3:40:10 methanol: Coomassie stock solution: ultrapure water: acetic acid). 12.0 g of Brilliant Blue R-250, 300 ml methanol and 60ml acetic acid were used to prepare Coomassie stock solution. The gel was incubated overnight in destaining solution (45:10:45 methanol: acetic acid: ultrapure water). Protein bands on the gel were visualised by a Image Quant GE Healthcare scanner system connected to a computer, and analysed by ImageQuant software with which densitometry was performed. Each experiment was done in triplicate.

Proteins of the isolated Staphylococcus phage were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [11 (link)]. Concentrated phage preparation (10¹⁰ PFU/mL approx.) was mixed with equal volume of 2X Laemmli buffer (Sigma, USA) and boiled for 10 minutes. A 25 μL sample mixture was loaded in each well along with 6 μL of broad range prestained protein ladder (Puregene, USA) in one well. The gel was run at 20 mA of constant current for 1 hour (approx.) till the tracking dye reached the bottom of the gel. The gel was finally stained with 0.25% Coomassie brilliant blue R-250 stain (Amresco, USA) for 4 hours and then suitably destained for best visibility of the protein bands with several changes of destaining solution and stored in 7% glacial acetic acid solution for photography. The gel was scanned in a scanner and documented.

Cell lysates were prepared by resuspending 5 x 10⁶ pelleted cells in 1X RIPA buffer (ThermoFisher) containing 5X Halt Protease Inhibitor (ThermoFisher) on ice for 15 min, followed by centrifugation at 14,000 x g for 10 min to clear the lysate. Protein concentration was quantified using the BCA Protein Quantification Kit (ThermoFisher). Samples were prepared by mixing 100 μg of lysate with 10 μL 2X Laemmli buffer (Sigma-Aldrich, USA) containing 1 μL beta-mercaptoethanol (Sigma-Aldrich). After adding Milli-Q water to 20 μL, samples were incubated at 95°C for 5 min.