TITLE: Selecting the correct transfer membrane for Western blot enhances detection of alpha-synuclein and tau proteins
AUTHORS: Gabriele Kaminski Schierle

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The ability to detect proteins by Western blot, particularly from tissue samples, can be hampered by the choice of membrane and whether a fixation step is used. Here we compare three transfer membranes, polyvinylidene difluoride (PVDF) 0.2 μm, PVDF 0.45 μm and nitrocellulose 0.45 μm for the detection of rat α-synuclein from tissue and recombinant human tau. For α-synuclein fixation of the membrane directly after transfer was imperative for detection of the protein and use of the PVDF 0.45 μm membrane gave the highest detection signal. For tau, the signal was highest on nitrocellulose 0.45 μm and fixation did not enhance the signal.</div></div>

[STEPS]
?. Protein samples were boiled for  in 1x  and ran on  in  using the  at 200 V for .
?. Three membranes were selected to test Western blot transfer and detection, PVDF 0.2 μm  PVDF 0.45 μm  and nitrocellulose 0.45 μm .
?. PVDF membranes were first activated with methanol by incubating for . Nitrocellulose membranes do not need to be activated first.
?. For detecting α-synuclein in tissue samples the membranes were fixed immediately after transfer with 4% paraformaldehyde (PFA), 0.1% glutaraldehyde in PBS for . Lack of fixation leads to poor/no α-synuclein detection and fixation with only 4% PFA also leads to lower detection than with the addition of 0.1% glutaraldehyde.
PFA and gluteraldehyde are toxic and should be handled in a chemical hood with appropriate PPE. The fixation solution should never be disposed of down the sink, but disposed of in a serparate container for disposal through correct waste systems.
?. Following disposal of the fixation solution into properly designated containers for removal, the membranes were blocked for  in 5% BSA in  + 0.05%  (PBS-T) at .
0 Room temperature
?. The membranes were then incubated either overnight or for  at  in primary antibody probing for rat α-synuclein 1:1000 dilution.
0 Room temperature
?. The membranes were washed three times for  in PBS-T.
?. The membranes were incubated for  at  in secondary antibody 1:4000
0 Room temperature
?. The membranes were washed five times for  in PBS-T.
?. The membranes were developed in  and imaged in a .
?. The gel was transferred onto each membrane using the  with  + 20%  at 30 V for .  The blot module was kept  and surrounded by ice to keep cool and prevent protein damage.
on ice
?. 
?. For recombinant human tau, fixation was not required and the membranes were instead immediately blocked for  in 5% BSA in PBS-T at .
0 Room temperature
?. The membranes were then incubated either overnight or for  at  in primary antibody probing for human tau 1:200 dilution .
0 Room temperature
?. The membranes were washed three times for  in PBS-T.
?. The membranes were incubated for  at  in secondary antibody, 1:4000 dilution
0 Room temperature
?. The membranes were washed five times for  in PBS-T.
?. The membranes were developed in Supersignal West Pico (#34580, ThermoFisher) and imaged in a .
?. 
?. This optimisation was originally performed by Dr Colin Hockings, a former postdoc in the Molecular Neuroscience Group.