Total protein concentrations were determined using the BCA kit (Pierce). Equal amounts of total protein extract were subjected to SDS–PAGE in 4–12% Bis–Tris gels (NuPAGE® Novex Bis-tris midi gel 15 wells, Invitrogen) and transferred to nitrocellulose membranes. Blocked membranes (5% milk in TBS–0.1% Tween-20) were incubated with primary antibodies: Crym [1 : 500, chicken IgY directed against AVGASRPDWRELDDE and affinity purified (AgroBio)], actin (1 : 2000, mouse, Sigma), HA (1 : 3000, mouse, Covance), Dopamine- and cAMP-regulated PhosphoProtein 32 kDa (DARPP-32, 1 : 3000, rabbit, Cell Signaling), α-tubulin (1 : 3000, mouse, Sigma); and washed three times with TBS–0.1% Tween-20 (TBST) for 10 min. Membranes were then labeled with secondary IgG-HRP antibodies raised against each corresponding primary antibody. After three washes with TBST, the membranes were incubated with ECL chimioluminescent reagent (Clarity Western ECL substrate; Bio-Rad or Immun-Star WesternC kit) according to the instructions of the supplier. Peroxydase activity was detected with camera system Fusion TX7 (Fisher Scientific). Normalization was done by densitometry analysis with Bio1D software.
Fusion tx7
Manufactured by Thermo Fisher Scientific
The Fusion TX7 is a high-performance liquid chromatography (HPLC) system designed for analytical and preparative applications. It features a modular design, allowing for the integration of various components to meet specific research or workflow requirements. The Fusion TX7 provides precise and reliable separation and detection of a wide range of analytes, supporting applications in fields such as pharmaceuticals, environmental analysis, and material science.
Automatically generated - may contain errors
Lab products found in correlation
Immun star westernc kit, by Bio-Rad (1 mentions)
Darpp 32, by Cell Signaling Technology (1 mentions)
Quantity one 1d image analysis software, by Bio-Rad (1 mentions)
Nupage novex bis tris midi gel 15 or 26 wells, by Thermo Fisher Scientific (1 mentions)
Clarity western ecl substrate, by GE Healthcare (1 mentions)
2 protocols using fusion tx7
1
Western Blot Quantification Protocol
2
Western Blot Protein Analysis Protocol
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Equal amounts of total protein extract (30 μg) were electrophoretically separated using SDS–PAGE in 4–12% Bis–Tris gels (NuPAGE® Novex Bis-tris midi gel 15 or 26 wells, Life Technologies, Carlsbad, USA) and transferred to nitrocellulose membranes. Blocked membranes (5% non-fat dry milk in TBS/0.1% Tween-20) were incubated with the primary antibodies (Table 1 ) overnight at 4 °C, and washed three times with TBS/0.1% Tween-20 (T-BST) for 10 min. Membranes were then labelled with secondary anti-IgG-HRP antibodies raised against each corresponding primary antibody. After three washes with T-BST, the membranes were incubated with ECL chemiluminescent reagent (Clarity Western ECL substrate; GE Healthcare, Little Chalfont, UK) according to the instructions of the supplier. Peroxidase activity was detected with the camera system Fusion TX7 (Fisher Scientific). Normalization was done by densitometry analysis with the Quantity One 1D image analysis software (version 4.4; Biorad, Hercules, CA, USA). The optical densities were normalized with respect to a housekeeping protein (GAPDH, actin or tubulin). A partition ratio was calculated and normalized with respect to the sample with the highest value defined as one.
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