Six male Dyt1 Ch2KO and eight CT littermates about 4 months of age, and seven male Dyt1 Ch2KO and six CT littermates at 13 months of age were used for Western blot analysis. The striata were dissected and homogenized in 200 μl of ice-cold lysis buffer, and the proteins were extracted in 1% Triton X-100-containing buffer and quantified as previously described (Yokoi et al., 2010 (link)). Protein samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Millipore Immobilon-FL transfer PVDF membranes. The membranes were blocked with LI-COR Odyssey blocking buffer and incubated at 4°C overnight with goat anti-ChAT antibody (EMD Millipore, AB144P; 1:1,000 dilution) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Santa Cruz, sc-25778; 1:1,000 dilution). LI-COR IRDye 800CW donkey anti-goat IgG (H+L) or LI-COR IRDye 680RD donkey anti-rabbit IgG (H+L) were used when appropriate at the dilution of 1:15,556. The signals were detected with an LI-COR Odyssey imaging system. The density of the corresponding protein band was normalized to those of GAPDH. Western blot analysis was performed in duplicate.
Immobilon fl transfer pvdf membranes
Manufactured by Merck Group
Immobilon-FL transfer PVDF membranes are a type of polyvinylidene fluoride (PVDF) membrane used for the transfer and detection of proteins in Western blotting applications. These membranes provide a high-binding capacity for protein samples and are designed to facilitate efficient protein transfer from polyacrylamide gels.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Amersham imager 600, by GE Healthcare (2 mentions)
Irdye 800cw donkey anti goat igg, by LI COR (1 mentions)
Ab144p, by Merck Group (1 mentions)
Irdye 680rd, by LI COR (1 mentions)
Sc 25778, by Santa Cruz Biotechnology (1 mentions)
Irdye 680rd donkey anti rabbit igg, by LI COR (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Irdye 800cw donkey anti goat igg h l, by LI COR (1 mentions)
Swine anti rabbit hrp conjugate, by Agilent Technologies (1 mentions)
Rabbit anti ha, by Merck Group (1 mentions)
Amersham imager, by Cytiva (1 mentions)
4 protocols using immobilon fl transfer pvdf membranes
1
Quantifying Cholinergic Markers in Dystonia Mouse Model
2
Cerebellar Protein Expression in Dyt1 KI Mice
3
Whole-Cell Lysate Western Blot Protocol
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Whole-cell lysates were used for western blot analysis. Cells were washed in PBS and resuspended in 1× Laemmli buffer (12 mM Tris-HCl pH 6.8, 0.4% SDS, 2% glycerol, 1% β-mercaptoethanol and 0.002% Bromophenol Blue) in PBS and incubated at 95°C for 5 min. Approximately 5×106 cells were loaded onto a 4% or 6% gel, resolved and then blotted (Bio-Rad blotting system) onto PVDF Immobilon®-FL transfer membranes (0.45 μm, Millipore) for 1 h at 100 V. Membranes were blocked in PBST plus 5% skimmed milk powder. The rabbit peroxidase anti-peroxidase soluble complex (PAP) was diluted 1:2000 in PBST plus 5% skimmed milk and incubated for 30 min at room temperature. The mouse-anti-EF1α (cat. no. sc-21758, Santa Cruz Biotechnology), rat-anti-HA and the rabbit-anti-HA antibodies (cat. no. ROAHAHA Roche and N6908-2ML, respectively, Sigma) were used 1:1000 in PBST plus 5% skimmed milk. Secondary antibodies were: swine anti-rabbit-IgG conjugated to horseradish peroxidase (HRP) (1:10,000, Dako) and rabbit anti-rat HRP conjugate (1:10,000, Dako), all in PBST plus 5% skimmed milk. After each incubation with the antibodies, the membranes were washed three times for 5 min in PBST and once for 5 min in PBS. SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) and Amersham™ Imager 600 (GE Healthcare Life Sciences) were used to visualize the protein bands on the blots.
4
Western Blot Analysis of Cellular Proteins
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Cell extracts were mixed with Laemmli buffer to a final concentration of 5 million cell-equivalents/15 μl. Either one million or five million cell-equivalents (depending on the experiment) were loaded per lane on an SDS-polyacrylamide gel. The gels were resolved by 80-120V and transferred (wet transfer, 100V for 1h) onto PVDF Immobilon- FL transfer membranes (0.45 μm, MILLIPORE), followed by blocking (5% milk in PBS + 0.1% Tween-20 (PBST)) for 1 h at room temperature. Primary antibodies were incubated for either 1 h at room temperature or overnight at 4°C. Secondary antibodies were applied for 1 h at room temperature. All used antibodies were diluted in blocking solution. The following antibodies were used: mouse anti-TAC102 (1:1000, [24 (link)]), rabbit anti-HA (1:1000, Sigma-Aldrich), rabbit anti-ATOM40 (1:10000, [61 (link)]), rabbit anti-mouse HRP-conjugated (1:10000, Dako) and swine anti-rabbit HRP-conjugate (1:10000, Dako), rabbit peroxidase anti-peroxidase soluble complex (PAP) (1:2000, for detection of the PTP tag). After antibody incubation, the membranes were washed three times with PBST. A final wash with PBS was performed prior to detection of the protein with the SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific). The Amersham Imager 600 (GE Healthcare) was used to visualize the protein bands on the blot.
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