Anti pgk1 antibody

To look at the protein level of wild type and mutants of Sbp1 in BG1805 construct, cells were first grown in SD ura-minimal media with glucose till 0.45–0.5 OD600 this was followed by pelleting and growing in SD ura- 2% galactose overnight. Cells were broken open using acid wash glass beads and 20microgram of total protein was loaded in 8% SDS polyacrylamide gel. The gel was transferred onto a nitrocellulose membrane using Bio-Rad wet transfer apparatus. Post transfer, the membrane was stained with Ponceau S to know the total protein present in each lane. The blot was washed and blocked using skimmed milk. PAP (1:5000, Sigma Aldrich cat# P1291) was used to detect over expressed Sbp1 and mutant proteins. Sbp1-GFP and its mutants protein level was looked at the same way as gal inducible Sbp1 except the use of anti-GFP anti body (1: 1000, BioLegend cat# 902602). For loading control, blot was stripped and put in anti - PGK1 antibody (1:1000, Abcam cat# AB113687).

Dot blot analysis was performed in a 96-well plate format using a Bio-Dot^® microfiltration apparatus (Bio-Rad, Herakles, CA, USA) for vacuum-transfer of the samples to nitrocellulose membranes (0.2 μm pore-size; Bio-Rad) pre-wetted with Tris-buffered saline (TBS; 20 mM Tris-HCl pH 7.5, 0.8% NaCl). Sample-loaded membranes were then blocked by incubation for 2 h at room temperature in TTBS (TBS supplemented with 0.1% Tween 20) containing 5% (w/v) bovine serum albumin (BSA). After blocking, membranes were incubated overnight at 4 °C with an anti-α-syn antibody (Santa Cruz Biotechnology Inc., Dallas, TX, USA; 1:200 dilution). An anti-Pgk1 antibody (Abcam, Cambridge, UK; 1:2000 dilution) was used as a loading control. Following washing with TTBS, membranes were incubated for 1 h at room temperature with IRDye-labeled goat anti-rabbit (for anti-α-syn) or goat anti-mouse (for anti-Pgk1) secondary antibodies (LI-COR Biosciences, Lincoln, NE, United States; 1:10000 dilution). Following washing with TTBS, membranes were dried and visualized with a Chemidoc MP Imaging System (Bio-Rad).