Standard SDS-PAGE protocol was used for analysis of ataxin 1 protein expression. Twenty to thirty milligrams of total protein were denatured, run on 7.5% Tris-HCl precast gel (Bio-Rad) and blotted on nitrocellulose membrane (Amersham Protran 0.45 μM, GE Healthcare). Membranes were blocked for 1 hour by 5% milk in PBST, and then incubated with primary antibodies overnight at 4 °C. After three times wash with PBST, the membrane was incubated with anti-mouse or anti-rabbit HRP conjugate antibody (Jackson ImmunoResearch Laboratories) for 30 minutes at room temperature. The membrane was soaked in SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and then expose to film. Primary antibodies were used as indicated: anti-ataxin 1 (A302-292A, 1:2000; Bethyl labs), anti-PolyQ (MAB1574, 1:10,000, Millipore), and anti-β-actin (1:10,000; Sigma).
Amersham protran 0.45 μm
Manufactured by GE Healthcare
Amersham Protran 0.45 μm is a nitrocellulose membrane designed for protein transfer and immobilization in western blotting applications. It has a pore size of 0.45 micrometers and is suitable for the transfer of proteins ranging from 10 to 200 kilodaltons.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Chemiluminescence substrate, by Bio-Rad (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Strep tactin hrp conjugate, by IBA Lifesciences (1 mentions)
Chemidoc mp imager, by Bio-Rad (1 mentions)
Anykd mini protean tgx gels, by Bio-Rad (1 mentions)
Anti rabbit igg hrp, by Cell Signaling Technology (1 mentions)
Anti mouse igg hrp, by Cell Signaling Technology (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
3 protocols using amersham protran 0.45 μm
1
SDS-PAGE Analysis of Ataxin 1 Protein
2
Western Blot Protein Detection
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Samples were mixed with 4x Laemmli buffer, denatured 3 min at 95°C and loaded on SDS-PAGE precast 4–15% gels (Mini-PROTEAN TGX gel, Bio-Rad) followed by electrophoresis at 200 V for 30 min in 1X TGS (Tris 2.5 mM, Glycine 19.2 mM, 0.01%SDS, pH8.3) buffer. After migration, proteins were electrotransferred to a nitrocellulose membrane (Amersham Protran 0.45 μm; GE Healthcare) during 1 h at 20 mA. The membranes were probed with rabbit Anti-RPL8 IgG antibodies (HPA050165; Sigma Prestige Antibodies, 1:1000) and then incubated with a secondary anti-rabbit IgG coupled to HRP (1:10,000). Alternatively, membranes were incubated with a Strep-Tactin-HRP conjugate (IBA Life Sciences, 2-1502-001, 1:2000). Membranes were incubated with a chemiluminescence substrate (Bio-Rad) and imaged on a ChemiDoc MP imager (Bio-Rad).
3
Western Blot Analysis of Viral Capsid Proteins
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The amount loaded per
lane was normalized
by OD600 readings (equivalent to 10 μL of culture
at OD600 = 20). The samples were run on Any kD Mini-PROTEAN
TGX gels (Bio-Rad) in Tris-glycine-SDS buffer at 150 V, 55 min, and
transferred onto nitrocellulose membranes (Amersham Protran 0.45 μm,
GE Healthcare) by wet transfer at 75 V, 60 min. The transfer buffer
was 1× SDS-PAGE buffer + 20% v/v methanol. Even protein transfer
was verified by staining the membrane with 0.1% w/v Ponceau S in 5%
v/v acetic acid. Membranes were blocked with 5% w/v skim milk in PBS
+ 0.05% v/v Tween 20 for >1 h at RT and incubated with primary
antibodies
overnight at 4 °C. Membranes were washed briefly with blocking
buffer and incubated with secondary antibodies for 90 min at RT. The
antibodies and dilutions used were as follows: rabbit anti-VP1 antiserum
1:2000, mouse anti-GFP monoclonal IgG (Cell Signaling Technology #2955)
1:2000; anti-rabbit IgG-HRP (Cell Signaling Technology #7074) 1:2000,
anti-mouse IgG-HRP (Cell Signaling Technology #7076) 1:2000. Rabbit
anti-VP1 antiserum was produced by Walter and Eliza Hall Institute
Antibody Services using wtVP1 expressed in E. coli and assembled into VLPs in vitro.18 (link) Blots were visualized with a Clarity Western ECL Substrate
(Bio-Rad #1705060) and imaged with a ChemiDoc MP Imaging System (Bio-Rad).
lane was normalized
by OD600 readings (equivalent to 10 μL of culture
at OD600 = 20). The samples were run on Any kD Mini-PROTEAN
TGX gels (Bio-Rad) in Tris-glycine-SDS buffer at 150 V, 55 min, and
transferred onto nitrocellulose membranes (Amersham Protran 0.45 μm,
GE Healthcare) by wet transfer at 75 V, 60 min. The transfer buffer
was 1× SDS-PAGE buffer + 20% v/v methanol. Even protein transfer
was verified by staining the membrane with 0.1% w/v Ponceau S in 5%
v/v acetic acid. Membranes were blocked with 5% w/v skim milk in PBS
+ 0.05% v/v Tween 20 for >1 h at RT and incubated with primary
antibodies
overnight at 4 °C. Membranes were washed briefly with blocking
buffer and incubated with secondary antibodies for 90 min at RT. The
antibodies and dilutions used were as follows: rabbit anti-VP1 antiserum
1:2000, mouse anti-GFP monoclonal IgG (Cell Signaling Technology #2955)
1:2000; anti-rabbit IgG-HRP (Cell Signaling Technology #7074) 1:2000,
anti-mouse IgG-HRP (Cell Signaling Technology #7076) 1:2000. Rabbit
anti-VP1 antiserum was produced by Walter and Eliza Hall Institute
Antibody Services using wtVP1 expressed in E. coli and assembled into VLPs in vitro.18 (link) Blots were visualized with a Clarity Western ECL Substrate
(Bio-Rad #1705060) and imaged with a ChemiDoc MP Imaging System (Bio-Rad).
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