| Reactivity | HuSpecies Glossary |
| Applications | WB |
| Concentration | 2 mg/ml |
| Description | Quality control test: 12.5% SDS-PAGE Stained with Coomassie Blue. Nuclear extract cell lysate (denatured) |
| Localization | Lung |
| Specificity | A-549 (human lung carcinoma) nuclear extract lysate (denatured) |
| 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 -80C. The protein concentration was determined by the method of Bradford. The lysate was adjusted to 2.5 mg/ml, and then mixed with 5X Sample Buffer to become final 2 mg/ml in 1X Sample Buffer. The lysate was heated at 95C for 5 min, and cooled rapidly. |
| Dilutions |
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| Application Notes | The lysate is ready to load on SDS-PAGE for Western blotting. |
| Storage | Store at -80C. Avoid freeze-thaw cycles. |
| Buffer | In ready-to-use 1X Sample Buffer (50 mM Tris-HCl, 2% SDS, 10% glycerol, 300 mM 2-mercaptoethanol, 0.01% Bromophenol blue). |
| Concentration | 2 mg/ml |
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