大肠杆菌细胞破碎方法（酶解和超声破碎）
大肠杆菌细胞破碎后液体的组成
Bacterial extracts roughly consist of protein (40-70%), nucleic acid (10-30%), polysacchartde (2-10%), and lipid (10-15%) The nucletc acid fraction can often cause high viscosity In addition to DNase treatment detailed in Section 3.1., step 4, this nucleic acid can also be removed from soluble protein solutions by precipitation with posmvely charged compounds, such as polyethyleneamme.
大肠杆菌细胞破碎后的液体中含蛋白质40-70%，核酸10-30%，多糖2-10%，脂类10-15%。该液体之所以显黏性是由细胞破碎后核酸释放出来造成的。可以加入DNase来消化掉核酸污染。
酶解破碎
1. Harvest the bacterial cells by centrtfugation at 1OOOg for 15 min at 4℃ and pour off the supernatant. Weigh the wet pellet This is easiest to do if you have preweighed the centrifuge tubes which can then be deducted from the total weight.
2. Add approx 3 mL of lysis buffer for each wet gram of bacterial cell pellet and resuspend. Add lysozyme to a concentration of 300 μg/mL and stir the suspension for 30 min at 4℃(see Notes 3 and 4).
3. Add deoxycholate to a concentratton of 1 mg/mL while stirring.
4. Place at room temperature, and add DNase 1 to a concentration of 10 mg/mL and MgCl2 to 10 mM. Stir suspension for a further 15 mm to remove the viscous nucleic acid (see Note 2).
5. Centrifuge the suspension at 10,000g for 15 mm at 4℃. Resuspend the pellet in lysis buffer to the same volume as the supernatant, and analyze altquots of both for the protein of interest on SDS-PAGE. If the bulk of a normally soluble protein is found in the insoluble pellet fraction, then inclusion bodies are likely to have formed.
超声破碎
1. Collect the cells by centrifugation at 1000g for 5 minutes. Remove the supernatant. Resuspend the cells in PBS and centrifuge again. Remove the PBS.
2. Resuspend the bacterial cell pellet in at least 10 cell volumes of ice-cold lysis buffer.
3. Use a probe- or cup-type sonicator to disrupt the bacterial cells with multiple short bursts of maximum intensity (e.g., four bursts, each 10-30 seconds). Between each burst, return the cells to an ice bath to keep the temperature from rising.
4. Centrifuge at 10,000g for 10 minutes at 4oC.
5. Carefully transfer the supernatant to a fresh tube. Keep on ice.
6. (Optional) Centrifuge the supernatant at 100,000g for 30 minutes at 4oC to remove aggregated proteins. Carefully transfer the supernatant to a fresh tube. Keep on ice.
7. The supernatant is now ready for preclearing (Immunoprecipitation, Preclearing the Lysate).
TROUBLESHOOTING
Problem: Protein antigens are not recognized by antibodies after lysis.
Solution: The most common problem with extracting any bacterial protein is the potential for denaturing the protein antigens during lysis. This is particularly true with the sonication procedure. If the antigen is not recognized by antibodies, try other lysis methods that are more gentle. One alternative method is to treat the bacteria with enzymes that will break down the bacterial cell wall (e.g., lysozyme treatment of Gram-negative bacteria) and then lyse the cells in detergent-based lysis buffers. A second approach is to switch to bacteria that harbor T7 genes, such as lysS. These bacteria constitutively make a viral lysozyme that removes the bulk of the cell wall, and the bacteria can be lysed as above by simple detergent treatment.
自 http://blog.bioon.net/user1/2081/archives/2009/229925.shtml