Reducing and non-reducing protein samples were separated by gel electrophoresis and transferred to nitrocellulose membranes using the Bolt SDS-PAGE system (Life Technologies). Membranes were blocked in 5% nonfat dry milk (NFDM) in TBS supplemented with 0.025% Tween-20 (TBS-T) for 1 h at RT, followed by incubation overnight at 4°C in primary antibody diluted in 5% bovine serum albumin/TBS-T. Primary antibodies used were as follows: Tau5 (1:1000), Tau13 (1:25,000), PHF1 (1:500) and CP13 (1:500) anti-tau antibodies (generously provided by P. Davies); TIA1 (1:500, Santa Cruz Biotechnology, sc-1751), total α-tubulin (1:5000), PABP (1:1000, abcam cat#ab21060), and DDX6 (1:500, Bethyl Labs cat#A300–460A). Membranes were then washed 3 times with TBS-T and incubated in HRP-conjugated secondary antibodies (Jackson ImmunoResearch) diluted in 1% NFDM/TBS-T at RT for 1 h. After incubation in secondary antibody, membranes were washed 3 times in TBS-T and developed using SuperSignal West Pico Chemilluminescent ECL substrate (ThermoFisher Scientific, cat #34080).
Supersignal west pico chemilluminescent ecl substrate
Manufactured by Thermo Fisher Scientific
SuperSignal West Pico Chemilluminescent ECL substrate is a laboratory product that generates chemiluminescent signals used for the detection of proteins in Western blot analysis.
Automatically generated - may contain errors
5 protocols using supersignal west pico chemilluminescent ecl substrate
1
Tau Protein Western Blot Analysis
2
Western Blot Analysis of Tau and Amyloid-β
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The homogenized lysate for western blot were collected from fresh frozen brain tissue with RIPA lysis buffer. Reducing and non-reducing protein samples were separated by gel electrophoresis and transferred to 0.2µm nitrocellulose membranes using the Bolt SDS-PAGE system (Life Technologies). Membranes were blocked in 5% nonfat dry milk (NFDM) in PBS supplemented with 0.025% Tween-20 (PBST) for 1 hour RT, followed by incubation overnight at 4°C in primary antibody diluted in 5% bovine serum albumin/PBST. Primary antibodies used were as follows: pTau217 (1:500) anti-tau antibody (rabbit, Thermo Scientific, Cat# 44744); 4G8, Anti-Amyloid β Antibody, clone W0-2, reactive to amyloid-β, aa 17–24 (Millipore Sigma, Cat# MABN10). Membranes were then washed 3 times with PBST and incubated in HRP-conjugated secondary antibodies (Jackson ImmunoResearch) diluted in 1% BSA/PBST at RT for 1 h. After incubation in secondary antibody, membranes were washed 3 times in PBST and developed using SuperSignal West Pico Chemilluminescent ECL substrate (ThermoFisher Scientific, cat# 34080).
3
Western Blot Analysis of Tau and Amyloid-β
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The homogenized lysate for western blot were collected from fresh frozen brain tissue with RIPA lysis buffer. Reducing and non-reducing protein samples were separated by gel electrophoresis and transferred to 0.2 μm nitrocellulose membranes using the Bolt SDS-PAGE system (Life Technologies). Membranes were blocked in 5% nonfat dry milk (NFDM) in PBS supplemented with 0.025% Tween-20 (PBST) for 1 h RT, followed by incubation overnight at 4°C in primary antibody diluted in 5% bovine serum albumin/PBST. Primary antibodies used were as follows: pTau217 (1, 500) anti-tau antibody (rabbit, Thermo Scientific, Cat# 44744); 4G8, Anti-Amyloid β Antibody, clone W0-2, reactive to amyloid-β, aa 17–24 (Millipore Sigma, Cat# MABN10). Membranes were then washed 3 times with PBST and incubated in HRP-conjugated secondary antibodies (Jackson ImmunoResearch) diluted in 1% BSA/PBST at RT for 1 h. After incubation in secondary antibody, membranes were washed 3 times in PBST and developed using SuperSignal West Pico Chemilluminescent ECL substrate (Thermo Fisher Scientific, cat# 34080).
4
Optimized Western Blot for Tau and Amyloid-β
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The homogenized lysate for western blot were collected from fresh frozen brain tissue with RIPA lysis buffer. Reducing and non-reducing protein samples were separated by gel electrophoresis and transferred to 0.2μm nitrocellulose membranes using the Bolt SDS-PAGE system (Life Technologies). Membranes were blocked in 5% nonfat dry milk (NFDM) in PBS supplemented with 0.025% Tween-20 (PBST) for 1 hour RT, followed by incubation overnight at 4°C in primary antibody diluted in 5% bovine serum albumin/PBST. Primary antibodies used were as follows: pTau217 (1:500) anti-tau antibody (rabbit, Thermo Scientific, Cat# 44744); 4G8, Anti-Amyloid β Antibody, clone W0-2, reactive to amyloid-β, aa 17-24 (Millipore Sigma, Cat# MABN10). Membranes were then washed 3 times with PBST and incubated in HRP-conjugated secondary antibodies (Jackson ImmunoResearch) diluted in 1% BSA/PBST at RT for 1 h. After incubation in secondary antibody, membranes were washed 3 times in PBST and developed using SuperSignal West Pico Chemilluminescent ECL substrate (ThermoFisher Scientific, cat# 34080).
5
Tau Protein Western Blot Analysis
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