Jun 04, 2020
Selecting the correct transfer membrane for Western blot enhances detection of alpha-synuclein and tau proteins
- 1University of Cambridge
Protocol Citation: Gabriele Kaminski Schierle 2020. Selecting the correct transfer membrane for Western blot enhances detection of alpha-synuclein and tau proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.bg6kjzcw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 04, 2020
Last Modified: June 04, 2020
Protocol Integer ID: 37804
Keywords: Western blot, gel transfer, alpha-synuclein, tau, PVDF, nitrocellulose, fixation,
Abstract
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.
Materials
STEP MATERIALS
NuPAGE™ LDS Sample Buffer (4X)Thermo FisherCatalog #NP0008
NuPAGE™ 4-12% Bis-Tris Protein Gels, 1.5 mm, 15-wellThermo FisherCatalog #NP0336PK2
NuPAGE™ MES SDS Running Buffer (20X)Thermo FisherCatalog #NP0002
Immobilon-PSQ PVDF Membrane 0.2um rollMillipore SigmaCatalog #ISEQ00005
Immobilon-P PVDF Membrane, 0.45um, rollMillipore SigmaCatalog #IPVH00010
Nitrocellulose Transfer MembraneAmersham BiosciencesCatalog #10600002
Methanol
NuPAGE™ transfer buffer Thermo Fisher ScientificCatalog #NP0006
PBS
Tween-20Sigma AldrichCatalog #P9416
Rabbit IgG horse radish peroxidase (HRP) linked Whole AbCatalog #GENA934-1ML
SuperSignal™ West Pico PLUS Chemiluminescent SubstrateThermo FisherCatalog #34579
Polyclonal Rabbit Anti-Human Tau Unconjugated Ig fractionAgilent TechnologiesCatalog #A002401-2
α-Synuclein (D37A6) XP® Rabbit mAbCell Signaling TechnologyCatalog #4179
Rabbit IgG horse radish peroxidase (HRP) linked Whole AbCatalog #GENA934-1ML
Protocol materials
NuPAGE™ 4-12% Bis-Tris Protein Gels, 1.5 mm, 15-wellThermo FisherCatalog #NP0336PK2
Materials, Step 1
Immobilon-PSQ PVDF Membrane 0.2um rollMerck MilliporeSigma (Sigma-Aldrich)Catalog #ISEQ00005
Materials, Step 2
Immobilon-P PVDF Membrane, 0.45um, rollMerck MilliporeSigma (Sigma-Aldrich)Catalog #IPVH00010
Materials, Step 2
NuPAGE™ transfer buffer Thermo Fisher ScientificCatalog #NP0006
Materials, Step 4
α-Synuclein (D37A6) XP® Rabbit mAbCell Signaling TechnologyCatalog #4179
Materials, Step 7
Polyclonal Rabbit Anti-Human Tau Unconjugated Ig fractionAgilent TechnologiesCatalog #A002401-2
Materials, Step 14
Nitrocellulose Transfer MembraneAmersham plcCatalog #10600002
Materials, Step 2
PBS
Materials, Step 6
SuperSignal™ West Pico PLUS Chemiluminescent SubstrateThermo FisherCatalog #34579
Materials, Step 11
Tween-20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P9416
Materials, Step 6
Rabbit IgG horse radish peroxidase (HRP) linked Whole AbCatalog #GENA934-1ML
In Materials, Materials and 2 steps
Methanol
Materials, Step 4
NuPAGE™ MES SDS Running Buffer (20X)Thermo FisherCatalog #NP0002
Materials, Step 1
NuPAGE™ LDS Sample Buffer (4X)Thermo FisherCatalog #NP0008
Materials, Step 1
Safety warnings
Follow safety guidelines and wear correct PPE when handling fication solution containing 4% paraformalsehyde and 0.1% gluteraldehyde
Protein samples were boiled for 00:03:00 in 1x
NuPAGE™ LDS Sample Buffer (4X)Sigma AldrichCatalog #NP0008
and ran on
NuPAGE™ 4-12% Bis-Tris Protein Gels, 1.5 mm, 15-wellSigma AldrichCatalog #NP0336PK2
in
NuPAGE™ MES SDS Running Buffer (20X)Sigma AldrichCatalog #NP0002
using the
Equipment
XCell SureLock Mini-Cell Electrophoresis System
NAME
Electrophoresis System
TYPE
Invitrogen
BRAND
EI0001
SKU
at 200 V for 00:30:00 .
Three membranes were selected to test Western blot transfer and detection, PVDF 0.2 μm
Immobilon-PSQ PVDF Membrane 0.2um rollSigma AldrichCatalog #ISEQ00005
PVDF 0.45 μm
Immobilon-P PVDF Membrane, 0.45um, rollSigma AldrichCatalog #IPVH00010
and nitrocellulose 0.45 μm
Nitrocellulose Transfer MembraneSigma AldrichCatalog #10600002
.
PVDF membranes were first activated with methanol by incubating for 00:03:00 . Nitrocellulose membranes do not need to be activated first.
The gel was transferred onto each membrane using the
Equipment
Invitrogen XCell II™ Blot Module
NAME
Western blot transfer module
TYPE
Invitrogen
BRAND
EI9051
SKU
with
NuPAGE™ transfer buffer Sigma AldrichCatalog #NP0006
+ 20%
MethanolSigma Aldrich
at 30 V for 01:30:00 . The blot module was kept On ice and surrounded by ice to keep cool and prevent protein damage.
Safety information
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.
For detecting α-synuclein in tissue samples the membranes were fixed immediately after transfer with 4% paraformaldehyde (PFA), 0.1% glutaraldehyde in PBS for 00:30:00 . 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.
Following disposal of the fixation solution into properly designated containers for removal, the membranes were blocked for 01:00:00 in 5% BSA in
PBSSigma Aldrich
+ 0.05%
Tween-20Sigma AldrichCatalog #P9416
(PBS-T) at Room temperature .
The membranes were then incubated either overnight or for 01:00:00 at Room temperature in primary antibody probing for rat α-synuclein 1:1000 dilution
α-Synuclein (D37A6) XP® Rabbit mAbSigma AldrichCatalog #4179
.
The membranes were washed three times for 00:05:00 in PBS-T.
The membranes were incubated for 01:00:00 at Room temperature in secondary antibody 1:4000
Rabbit IgG horse radish peroxidase (HRP) linked Whole AbSigma AldrichCatalog #GENA934-1ML
The membranes were washed five times for 00:05:00 in PBS-T.
The membranes were developed in
SuperSignal™ West Pico PLUS Chemiluminescent SubstrateSigma AldrichCatalog #34579
and imaged in a
Equipment
G:Box Chemi
NAME
Imaging system
TYPE
Syngene
BRAND
XX6
SKU
.
Expected result
The best signal for detection of alpha-synuclein from rat tissue was obtained on PVDF 0.45 μm with fixation in 4% PFA and 0.1% glutaraldehyde.
For recombinant human tau, fixation was not required and the membranes were instead immediately blocked for 01:00:00 in 5% BSA in PBS-T at Room temperature .
The membranes were then incubated either overnight or for 01:00:00 at Room temperature in primary antibody probing for human tau 1:200 dilution
Polyclonal Rabbit Anti-Human Tau Unconjugated Ig fractionSigma AldrichCatalog #A002401-2
.
The membranes were washed three times for 00:05:00 in PBS-T.
The membranes were incubated for 01:00:00 at Room temperature in secondary antibody, 1:4000 dilution
Rabbit IgG horse radish peroxidase (HRP) linked Whole AbSigma AldrichCatalog #GENA934-1ML
The membranes were washed five times for 00:05:00 in PBS-T.
The membranes were developed in Supersignal West Pico (#34580, ThermoFisher) and imaged in a
Equipment
G:Box Chemi
NAME
Imaging system
TYPE
Syngene
BRAND
XX6
SKU
.
Expected result
The best signal for recombinant Tau was obtained on nitrocellulose 0.45 μm.
This optimisation was originally performed by Dr Colin Hockings, a former postdoc in the Molecular Neuroscience Group.