• 我要登录|
  • 免费注册
    |
  • 我的丁香通
    • 企业机构:
    • 成为企业机构
    • 个人用户:
    • 个人中心
  • 移动端
    移动端
丁香通 logo丁香实验_LOGO
搜实验

    大家都在搜

      大家都在搜

        0 人通过求购买到了急需的产品
        免费发布求购
        发布求购
        点赞
        收藏
        wx-share
        分享

        Silver Staining SDS PAGE Gels

        互联网

        1432

         

        We currently use the SilverQuest Silver stain kit from Invitrogen. This method is faster than the method listed below. The rapid method works well for most applications. Click here for instructions to the SilverQuest kit


        Non-Kit based Silver staining Method:

        Hahn Lab: Kyle Coachman, Jeff Ranish, Steve Hahn

        last modified 5/13/02

        Materials Needed:

        1. Silver Nitrate
        2. 1% Citric Acid: 100 ml of DI water + 1 g of Citric Acid
        3. 30 % NaOH(7.5 M): 100 ml of DI water + 30 g of NaOH
        4. 14.8 M Ammonium Hydroxide
        5. 38% Formaldehyde
        6. Ultra-pure water, use this for all steps and reagents
        7. 50% Aqueous Glutaraldehyde (optional)
        8. Glass tray or Novex Stain Ease Gel tray. If using glass, make sure to clean well with soap and DI water. If using Novex tray, use only trays designated for use with silver staining � do not use trays which have been used for coomassie staining

         

         Method:

        1. Make 7% Acetic Acid: 186 ml of water + 14 ml of acetic acid
        2. Make 50% Methanol: 200 ml of water + 200 ml of methanol. Optional � for extra fixation/crosslinking add 240 µl of 50% glutaraldehyde to the 50% methanol (makes solution 0.03% glutaraldehyde).
        3. Soak gel in 7% acetic acid for 7 minutes.
        4. Soak gel in 200 ml of 50% methanol for 20 minutes.
        5. Soak gel in 200 ml of 50% methanol for 20 minutes.
        6. Prepare Solution A: 0.8 g of silver nitrate + 4 ml of water
        7. Rinse gel in ~200 ml water for 10 minutes
        8. Rinse gel in ~200 ml water for 10 minutes

          Note: Steps 7 and 8 are very important for the NuPAGE gels if you skip these steps or do not rinse the gel for long enough the gel will develop too quickly and have significantly more background.

        9. 5 minutes before end of final water rinse prepare solution B: 21 ml of water + 250 ul of 30% NaOH (to make 0.36%) + 1.4 ml of 14.8M ammonium hydroxide
        10. To make staining solution: add solution A to solution B dropwise while stirring then add 76 ml of water.
        11. Soak gel in the staining solution for 15 minutes
        12. Rinse gel in ~200 ml water for 5 minutes
        13. Rinse gel in ~200 ml water for 5 minutes
        14. Make developing solution: 200 ml of water + 1 ml of 1% citric acid + 100 ul of 37% formaldehyde
        15. Soak gel in developing solution until bands are visible usually 2 to 15 minutes
        16. Stop development by rinsing gel with 3 changes of ~200 ml water

         The sensitivity of this method should be in the 10ng/band range.

         

        ad image
        提问
        扫一扫
        丁香实验小程序二维码
        实验小助手
        丁香实验公众号二维码
        扫码领资料
        反馈
        TOP
        打开小程序