【求助】求质谱兼容的银染方法

最新回复

• 箭头儿 (2014-4-12 10:08:06)

  
  我有一篇文献,是和质谱兼容的银染方法,我用文献中提到的第二种方法可以检测到5ng的蛋白,但要求所需的试剂是进口的才行,最少也要求硝酸银是进口的,因为我们开始用国产试剂最低只能检测到100ng,把硝酸银改为进口的后就可以到5ng了.不过还没上质谱,所以不知道质谱的效果如何.希望能对你有所帮助.
  因文件太大,不能上传,只能把操作步骤上传给你了
  EMBL Silver Staining Protocol
  Procedure
  1. Fix gel in 50% MeOH, 5% HAc for 20 mins.
  2. Wash gel in 50% MeOH for 10 mins.
  3. Wash gel in H2O for 2 hrs.
  Additional washing over night will reduce background
  staining.
  4. Sensitize gel in 0.02% Na2S2O3 for 1 min.
  5. Wash gel in H2O for 1 min.
  6. Wash gel in H2O for 1 min.
  7. Incubate gel in cold 0.1% AgNO3 for 20 mins.
  8. Wash gel in H2O for 1 min.
  9. Change gel chamber
  10. Wash the gel in H2O for 1 min.
  11. Develop gel in 0.04% formalin, 2% Na2CO3
  Observe the color and change solution when the developer
  turns yellow. Terminate when the staining is sufficient.
  12. Terminate staining in 5% HAc
  Change the solution a couple of times
  13. Leave the gel at 4ºC in 1% HAc
  Blum Silver Staining Protocol (modified)
  Procedure
  1. Fix gel in 40% EtOH, 10% HAc for 1 hr.
  2. Wash gel in 30% EtOH, 2 x 20 mins.
  3. Wash gel in H2O for 20 mins.
  4. Sensitize gel in 0.02% Na2S2O3 for 1 min.
  5. Wash gel in H2O, 3 x 20 secs.
  6. Incubate gel in cold 0.1% AgNO3, 20 mins. at 4ºC
  7. Wash gel in H2O, 3 x 20 secs.
  8. Change gel chamber
  9. Wash the gel in H2O for 1 min.
  10. Develop gel in 3% Na2CO3, 0.05% formalin
  Observe the color and change solution when the developer
  turns yellow. Terminate when the staining is sufficient.
  11. Wash the gel in H2O for 20 secs.
  12. Terminate staining in 5% HAc
  13. Wash the gel in H2O, 3 x 10 mins.
  14. Leave the gel at 4ºC in 1% HAc
  Vorum Silver Staining Protocol
  Procedure
  1. Fix gel in 50% MeOH, 12% HAc, 0.05% formalin for 2 hrs.
  or overnight
  2. Wash gel in 35% EtOH for 20 mins.
  3. Wash gel in 35% EtOH for 20 mins.
  4. Wash gel in 35% EtOH for 20 mins.
  5. Sensitize gel in 0.02% Na2S2O3 for 2 mins.
  6. Wash gel in H2O for 5 mins.
  7. Wash gel in H2O for 5 mins.
  8. Wash gel in H2O for 5 mins.
  9. Stain gel in 0.2% AgNO3, 0.076% formalin for 20 mins.
  10. Wash gel in H2O for 1 min.
  11. Wash gel in H2O for 1 min.
  12. Develop gel in 6% Na2CO3, 0.05% formalin, 0.0004%
  Na2S2O3
  13. Stop staining in 50% MeOH, 12% HAc for 5 mins.
  14. Leave the gel at 4ºC in 1% HAc

• yes4 (2014-4-12 10:08:32)

  谢谢版主,问下你给了三种方法,哪一种好呢?

• yes4 (2014-4-12 10:08:49)

  
  另外,能不能告诉我一下文献的名字,我查下看看~

• 箭头儿 (2014-4-12 10:09:13)

  只要不使用戊二醛，任何一种方法都行。

• 箭头儿 (2014-4-12 10:09:36)

  用的是第二种方法。