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[求助]
β半乳糖苷酶的酶活定量实验步骤求助
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请问各位大侠： 有没有谁做过β半乳糖苷酶的酶活定量实验啊？我想做一下这个酶的酶活定量十余年，可是不知道怎么做啊，要用什么药剂，要具体步骤，急，求助！~~~ 谢谢啦 |
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【答案】应助回帖
★ ★
感谢参与，应助指数 +1
zhongjin1988(gyesang代发): 金币+2, 鼓励回帖应助！ 2013-06-03 12:43:25
感谢参与，应助指数 +1
zhongjin1988(gyesang代发): 金币+2, 鼓励回帖应助！ 2013-06-03 12:43:25
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Cell Staining for Senescence-Associated â-Galactosidase (SA-â-Gal) Activity Posted on Monday, October 27, 2003 Description Cell Staining for Senescence-Associated â-Galactosidase (SA-â-Gal) Activity Procedure 1. Carefully remove the growth media from the cell culture. 2. Add an equal volume of PBS and gently mix the PBS solution around to wash out the cell culture media. 3. Repeat the PBS wash of the cells two more times. 4. Discard the PBS wash solution. 5. Add Fixative Solution to the cells and incubate at room temperature for 3 to 5 min (see Hint #2). 6. Wash cells as before in PBS two times. 7. Add between 1 to 2 ml of Staining Solution per 35 mm culture dish or cover slide. 8. Incubate for at least 2 hr at 37°C (do not incubate in a CO2 incubator; see Hint #3). Recipes Staining Solution 1 ml of 100 mM K3Fe(CN)6 (final concentration 5 mM) 40 ìl of 1 M MgCl2 (final concentration 2 mM) *Add just before use 600 ìl of 5 M NaCl (final concentration 150 mM) Prepare Staining Solution the day of experiment 4 ml of Citric Acid/Phosphate Buffer (final concentration 30 mM) 1 ml of 100 mM K4Fe(CN)6 (final concentration 5 mM) 1 ml of X-Gal Solution* (final concentration 1 mg/ml) Cell Fixative in PBS 2% (v/v) Formaldehyde 0.2% (w/v) Glutaraldehyde 1 M MgCl2 5 M NaCl X-Gal Solution in DMF (CAUTION! see Hint #1) 20 mg/ml X-Gal (5-bromo-4-chloro-3-indoly Â-D-galactopyranoside) Store -20°C X-Gal is not stable in water and should be added to the Staining Solution just before use 100 mM Potassium Ferricyanide 100 mM Potassium Ferricyanide (K3Fe(CN)6) CAUTION see Hint #1 100 mM Potassium Ferrocyanide 100 mM Potassium Ferrocyanide (K4Fe(CN)6) CAUTION see Hint #1 Citric Acid/Phosphate Buffer 0.1 M Citric Acid 0.2 M Sodium Phosphate Dibasic (Na2HPO4) Proper pH is VERY important pH 6.0 PBS pH 7.2 2.7 mM KCl 4.3 mM Sodium Phosphate Dibasic (Na2HPO4) 1.8 mM Potassium Phosphate Monobasic (KH2PO4) 137 mM NaCl Supplies Tips 1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions. 2. Cells can also be fixed in 3% (v/v) Formaldehyde in PBS; however the Fixative Solution preserves morphology better. 3. A blue color is visible within 2 hr; however, staining is maximum at between 12 to 16 hr. Blue stain is indicative of senescence-associated â-galactosidase activity. |
2楼2013-06-03 04:39:47
