|Product Overview :||Yeast Nuclei Isolation kit enables fast and easy purification of nuclei from yeast cells, utilizing yeast cell wall lysis and homogenization.|
|Applications :||DNA-Protein interaction, RNA-Protein interaction and Protein-Protein interaction studies
DNase I footprinting analysis, Enzymatic Assays and Pull-down assay
Western blot and ELISA
|Storage :||Store kit at -20centigrade. Warm Buffers A and B to room temperature (RT) before use. Read the entire protocol before performing the assay.|
|Kit Components :||• Buffer A
• Buffer B
• 1 M DTT
• Lysis Enzyme Mix
• Buffer N
• Protease Inhibitor Cocktail (Lyophilized)
|Materials Required but Not Supplied :||• Media to grow yeast cells
• Dounce Tissue homogenizer
• Microscope to visualize nucleus
• Refrigerated centrifuge
|Features & Benefits :||Simple, rapid and easy method to purify yeast nuclei.|
|Preparation :||• Buffer A: Store at 4centigrade. Warm to RT & add DTT to final conc. of 10 mM prior to use to the desired volume of buffer.
• Buffer B: Store at 4centigrade. Warm to RT & add Lysis Enzyme Mix to the desired volume of Buffer (5 µl/ml of Buffer) just before use.
• Lysis Enzyme Mix: Aliquot and store at -20centigrade.
• Buffer N: Store at 4centigrade. Keep on ice while in use. Add Protease Inhibitor Cocktail (1:1000) before use or as needed.
• Protease Inhibitor Cocktail: Resuspend Protease Inhibitor Cocktail in 250 µl of DMSO. Store at -20centigrade.
|Separation Protocol :||The described procedure is for small-scale isolation (10-20 ml) for total OD~20. For a large-scale preparation (total OD~200), calculate the reagent volumes accordingly.
1. Yeast Culture: Grow yeast cells in appropriate media overnight at 30centigrade, shaking at 200 rpm. For temperature-sensitive mutants use the appropriate temperature. When cells are in log phase, determine the OD of the culture at 600 nm. Multiply the OD by the total volume of the culture (ml) to calculate the total OD.
2. Nuclei Isolation:
2.1 Centrifuge the yeast culture at 3,000 x g at RT for 5 min. and discard the supernatant. Wash the cells by resuspending in 2 volumes of ultrapure water. Resuspend the cell pellet in 1 ml of Buffer A containing DTT and incubate for 10 min. at 30centigrade with gentle shaking. Centrifuge at 1,500 x g at RT for 5 min. and discard the supernatant.
2.2 Resuspend the cell pellet in 1 ml of Buffer B. Aliquot 10 µl suspension in separate glass tube (Control). Add 10 µl Lysis Enzyme Mix to the remaining cell suspension and incubate for 10-15 min. at 30centigrade in shaking incubator. Aliquot 10 µl of suspension again in another glass tube.
Note: To check the efficiency of spheroplast formation, add 990 µl of water to 10 µl aliquot from step 2.2 (Control & with Lysis Enzyme Mix). Measure OD at 600 nm. Incubation should continue until the OD of the sample is decreased 30-40% after adding Lysis Enzyme Mix compared to Control.
2.3 Centrifuge spheroplasts at 1,500 X g for 5 min. and discard the supernatant. From this step onwards, keep the tubes on ice.
Resuspend the spheroplast pellet in 1 ml of Buffer N with protease inhibitor cocktail. Transfer the suspension to a Glass Dounce homogenizer (not provided) and stroke 3-5 times on ice. Shake the suspension for 30 min. at RT. Centrifuge at 1,500 x g for 5 min at 4centigrade to remove the debris. Collect the supernatant. Centrifuge at 20,000 x g for 10 min. at 4centigrade to pellet the nuclei. Discard the supernatant and resuspend the nuclei pellet in Buffer N. Determine the protein concentration and adjust the desired protein concentration by Buffer N accordingly. Check the quality of nuclei under light microscope or with DAPI by adding 4 µl nuclei to 4 µl of DAPI (1µg/ml) and view under the fluorescence microscope.
Note: Application Based Storage Conditions: For intact nuclei, snap freeze in liquid nitrogen and store frozen nuclei at -80centigrade.
For gel loading purposes, nuclei can be stored in Lysis Buffer or SDS PAGE loading dye (Not provided).
|Sample Type :||Yeast cell culture|