Experiments on the preparation of yeast extracts
Experiments on the preparation of yeast extracts
Source:Laboratory Guide to Proteins and Proteomics
Operation method
basic program
Principle
Yeast cells can be lysed by a variety of methods, including autolysis, pressure fragmentation (e.g., French pressurized tanks), milling (oscillating glass beads), and enzymatic lysis (e.g., Zymolase). Oscillatory glass bead grinding, discussed in this protocol, is one of the easiest methods. This is effective for both small volumes (e.g., <1 mL) and several liter volumes. Cell fragmentation is typically >95% as detected by phase contrast microscopy.
Materials and Instruments
Freshly cultured yeast cells Move ① Centrifuge the cells for 3mm on a microcentrifuge to collect the cells. ② Remove the supernatant, resuspend the cells with PBS and centrifuge for another 3 min. ③ Remove the supernatant, estimate the volume of cell sediment, add 3 times the volume of ice bath lysis buffer to the cell sediment, suspend and place on ice. solution, suspend and place on ice. Add an equal volume of pre-cooled glass beads. ⑤ Vigorously shake the suspension for 30s. ⑥ Repeat step ⑤ and observe under a phase contrast microscope until most of the yeast cells are lysed. ⑦ Centrifuge the suspension in a small centrifuge for 5 min at 4°C. ⑧ Carefully pour out the supernatant and transfer to another tube and set aside on ice. Caveat Equipment pretreatment: glass beads were washed twice in 1 mol/L HCl and then twice in lysis buffer. The washed glass beads were soaked in a small amount of lysis buffer stored at 4°C. For more product details, please visit Aladdin Scientific website.
Lysis buffer (RIPA buffer) PBS
Pre-cooled glass beads