Total proteins from T lymphocytes or lung samples were prepared according to the protein extract kit instructions (Beyotime, China), after which the concentration of protein was determined using a BCA protein assay kit (Pierce, USA). The protein extracts were fractionated on a 10 % polyacrylamide-sodium dodecyl sulfate (SDS) gel and then transferred to a nitrocellulose membrane. The membrane was blocked with 5% (w/v) nonfat milk in Tris-buffered saline containing 0.05 % Tween-20, after which the membranes were then incubated overnight at 4 °C with an anti-phospho-P65 antibody (Cell Signaling Technology, USA, #3039), anti-phospho-Stat5 antibody (Cell Signaling Technology, USA, #9356), anti-phospho-Stat3 antibody (Cell Signaling Technology, USA,#9138), or β-actin antibody (Cell Signaling Technology, USA,#3700). The densitometric analyses of bands were performed by the use of an Imager system (BIO-RAD).
Protein extract kit
Manufactured by Beyotime
Sourced in China
The Protein Extract Kit is a laboratory equipment designed to extract and purify proteins from various biological samples. It provides a simple and efficient method for isolating proteins without the need for complex and time-consuming procedures.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho p65 antibody, by Cell Signaling Technology (1 mentions)
Anti phospho stat5 antibody, by Cell Signaling Technology (1 mentions)
Anti phospho stat3 antibody, by Cell Signaling Technology (1 mentions)
β actin antibody, by Cell Signaling Technology (1 mentions)
X ray film, by Kodak (1 mentions)
Molecular imager, by Bio-Rad (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
3 protocols using protein extract kit
1
Western Blot Analysis of Signaling Proteins
2
Protein Extraction and Western Blotting
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Total proteins from frozen liver samples were prepared according to the method described by the protein extract kit (Beyotime, Shanghai, China). The total protein concentration was determined using the BCA protein assay kit (Pierce, USA). Protein extracts were fractionated on 12% polyacrylamide-sodium dodecyl sulfate (SDS) gel and then transferred to nitrocellulose membrane. The membrane was blocked with 5%(w/v) nonfat milk in Tris-buffered saline (TBS) containing 0.05% tween-20, and then the membrane was incubated with primary antibody overnight at 4°C, followed by incubation with secondary antibody. Antibody binding was visualized with an ECL chemiluminescence system (Pierce, USA) and short exposure of the membrane to X-ray films (Kodak, Japan).
3
Protein Expression Analysis in Mice Skin
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The total protein was extracted from the mice back skin with protein extract kit (Beyotime, Shanghai, China) according to the instructions. The protein quantification was performed using BCA protein assay kit (Beyotime, Shanghai, China). The samples (40 μg per sample) were separated by polyacrylamide gel electrophoresis. Subsequently, the protein was transferred to polyvinylidene fluoride membrane and blocked with 5% skim milk. The protein band was determined with Bio-Rad Molecular Imager (Bio-rad, Hercules, CA, USA) after the incubation of the first antibody Collagen I and GAPDH (Cloud-Clone Corp, China) and the secondary antibody: anti-rabbit IgG (Cloud-Clone Corp, China). Protein expression was quantified by Image J software (NIH, Bethesda, MD, USA).
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