After washing with PBS, cells were lysed with 2 × SDS loading buffer [100 mM Tris-Cl (pH 6.8), 4% SDS, 0.2% bromophenol blue, 20% glycerol, 10% 2-mercaptoethanol] and then boiled for 10 min. Proteins were separated by SDS PAGE and transferred to a nitrocellulose membrane. The membranes were incubated with blocking buffer (PBS, 5% milk, 0.05% Tween) for 1 h and then with primary antibody diluted in the blocking buffer. After three washes with PBST (PBS, 0.05% Tween), the membranes were incubated with secondary antibody. After three washes with PBST, the membrane was visualized by Western Lightning Plus-ECL substrate (PerkinElmer; NEL10500) or by an Odyssey CLx Imaging System. The protein bands were quantified by densitometry with ImageJ if necessary.
Odyssey clx imaging system
Manufactured by PerkinElmer
The Odyssey CLx Imaging System is a high-performance, near-infrared fluorescence imaging system designed for a wide range of applications in life science research. It provides sensitive and quantitative detection of fluorescent signals on a variety of sample types, including Western blots, gels, and microplates.
Automatically generated - may contain errors
4 protocols using odyssey clx imaging system
1
Western Blot Analysis Protocol
2
Quantitative Western Blot Analysis
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For western blot analysis, samples were loaded into 4%–15% Mini-PROTEAN TGX Precast gels (Bio-Rad), together with a fluorescent protein ladder (LI-COR ref. 928-60000). Proteins were separated by electrophoresis in SDS running buffer for ∼45min at 200V. Subsequently, proteins were transferred on a nitrocellulose membrane at 4°C overnight at 23V. The membrane was blocked for 1h at room temperature with PBS supplemented with 10% non-fat skimmed milk and 0.1% Tween. The membrane was then incubated for 90min at room temperature with primary antibodies (see Table S4 ) diluted at the appropriate concentration in PBS supplemented with 5% non-fat skimmed milk and 0.1% Tween. The membrane was washed 4 times with PBS supplemented with 0.1% Tween, and incubated for 2h at room temperature with fluorescently labeled (LI-COR IRDye) or HRP-conjugated (GE Healthcare) secondary antibodies diluted in PBS supplemented with 5% non-fat skimmed milk and 0.1% Tween. The membrane was finally washed 4 times with PBS supplemented with 0.1% Tween. Proteins were visualized using the LI-COR Odyssey CLx imaging system (fluorescence) or detected on film by chemiluminescence (PerkinElmer ECL kit). Western blot signal was quantified using the LI-COR Image Studio software by measuring the fluorescence intensity of appropriate protein bands.
3
Quantitative Western Blot Analysis
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After washing with PBS, cells were lysed with 2× SDS loading buffer (100 mM Tris-Cl [pH 6.8], 4% SDS, 0.2% bromophenol blue, 20% glycerol, 10% 2-mercaptoethanol) and then boiled for 5 min. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were incubated with blocking buffer (PBS, 5% milk, 0.05% Tween) for 1 hour and then with a primary antibody diluted in blocking buffer. After three washes with PBST (PBS, 0.05% Tween), the membranes were incubated with a secondary antibody. After three washes with PBST, the membrane was visualized by Western Lightning Plus-ECL substrate (PerkinElmer, NEL10500) or by an Odyssey CLx Imaging System. The protein bands were quantified by densitometry with ImageJ as necessary.
4
Western Blot Protein Quantification
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After washing with phosphate buffered saline (PBS) , cells were lysed with 2× SDS loading buffer [100 mM Tris-Cl (pH 6.8), 4% SDS, 0.2% bromophenol blue, 20% glycerol, and 10% 2-mercaptoethanol] and then boiled for 10 min. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were incubated with blocking buffer (PBS, 5% milk, and 0.05% Tween) for 1 h and then with primary antibody diluted in the blocking buffer. After three washes with PBST (PBS, 0.05% Tween), the membranes were incubated with a secondary antibody. After three washes with PBST, the membrane was visualized by Western Lightning Plus-ECL substrate (PerkinElmer; NEL10500) or by an Odyssey CLx Imaging System. The protein bands were quantified by densitometry with ImageJ if necessary.
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