A-549 (human lung carcinoma) nuclear extract lysate (non-denatured)
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Specification
Product Description
Nuclear extract cell lysate (non-denatured).
Tissue
Lung
Host
Human
Preparation Method
Nuclear extract was prepared by using a modified protocol of Dignam et al. Cells were Harvested and homogenized in Buffer A, and then centrifugated at 25,000 g for 20 minutes to remove cytoplasm and pellet the nuclei. The pellet was re-suspended in Buffer C, and then the suspensions were centrifuged to collect nuclear extract. The supernatant was dialyzed against Buffer D. The dialysate was then centrifuged, divided into aliquots, and stored at -80°C. The protein concentration was determined by the method of Bradford (Bio-Rad protein assay, microplate standard assay). The lysate was adjusted to 2 mg/ml.
Lysis Buffer
Buffer A: 10mM HEPES pH 7.9, 1.5mM MgCl2, 10mM KCl, 0.5 mM DTT.
Buffer C: 20mM HEPES pH 7.9, 25%(v/v) Glycerol , 0.42M NaCl , 1.5mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT & 0.5 mM PMSF.
Buffer D : 20mM HEPES pH 7.9, 20%(v/v) glycerol, 50mM KCl, 0.2 mM EDTA, 0.5 mM DTT & 0.5 mM PMSF.Quality Control Testing
12.5% SDS-PAGE Stained with Coomassie Blue.
SDS-PAGE Gel
Recommend Usage
Use it directly for immuno-precipitation, or heat lysate with SDS gel loading buffer to 95°C for 5 minutes followed by rapid cooling for western blot application. If dissociating conditions are required, add reducing agent prior to heating.
Storage Buffer
In Buffer D.
Storage Instruction
Store at -80°C. Aliquot to avoid repeated freezing and thawing.
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Applications
Western Blot
Immunoprecipitation
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