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지식나눔

지식나눔

2-DE..

2006-03-31 org.kosen.entty.User@7dfee1c8
  • 1
2-DE를 했는데요... pH가 낮고 분자량이 큰부분에 horizontal streaking이 심하게 일어납니다.. 왜 이렇게 streaking이 일어날까요?? 조언 좀 부탁드립니다..
  • 2-DE
지식의 출발은 질문, 모든 지식의 완성은 답변! 
각 분야 한인연구자와 현업 전문가분들의 답변을 기다립니다.
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    김지수님의 답변

    2006-03-31
    • 0
    sample prep과정을 체크해 보세요 desalting은 잘 되었죠? salt가 streaking을 일으키는 주된 원인인건 아시죠? 3kD cut-off나 TCA-acetone precipitation을 이용하시죠? 저분자량 단백질을 보시려면 고분자량 단백질 제거 kit를 사용해 보시는것도 괜찮고요. 단백질 양을 줄여보는 것도 한 가지 방법이 되겠네요 너무 일반적인 것만 적어서 도움이 되셨는지 모르겠네요.. 아래 사항도 참고해 보셔요 1. Problem: Horizontal streaking across entire gel 2. Likely Cause(s): Proteins not properly and stably solubilized 3. Recommended Solution(s): (1)Ensure that all proteins are completely solubilized by using a suitably strong chaotropic extraction reagent. The concentrations of urea, thiourea, detergents (e.g., CHAPS, ASB-14), carrier ampholytes, and reducing agents (DTT or TBP) are also critical. Every sample type typically requires a new sample preparation method. A good starting point for sample preparation includes the following standard buffer solutions: 8–9 M urea, 4% (w/v) CHAPS, 2 mM TBP or 1% (w/v) DTT, 40 mM Tris, 0.2% (w/v) Bio-Lyte (ampholyte), pH 3–10 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 2 mM TBP or 1% (w/v) DTT, 40 mM Tris, 0.2% (w/v) Bio-Lyte (ampholyte), pH 3–10 (2)Allow sufficient time for full denaturation and solubilization. It is often beneficial to allow the sample to sit at room temperature in the solubilization solution for one hour before applying to IEF. (3)Remove insoluble protein complexes by centrifugation (>10,000 x g) prior to isoelectric focusing.
    답변수정
    sample prep과정을 체크해 보세요 desalting은 잘 되었죠? salt가 streaking을 일으키는 주된 원인인건 아시죠? 3kD cut-off나 TCA-acetone precipitation을 이용하시죠? 저분자량 단백질을 보시려면 고분자량 단백질 제거 kit를 사용해 보시는것도 괜찮고요. 단백질 양을 줄여보는 것도 한 가지 방법이 되겠네요 너무 일반적인 것만 적어서 도움이 되셨는지 모르겠네요.. 아래 사항도 참고해 보셔요 1. Problem: Horizontal streaking across entire gel 2. Likely Cause(s): Proteins not properly and stably solubilized 3. Recommended Solution(s): (1)Ensure that all proteins are completely solubilized by using a suitably strong chaotropic extraction reagent. The concentrations of urea, thiourea, detergents (e.g., CHAPS, ASB-14), carrier ampholytes, and reducing agents (DTT or TBP) are also critical. Every sample type typically requires a new sample preparation method. A good starting point for sample preparation includes the following standard buffer solutions: 8–9 M urea, 4% (w/v) CHAPS, 2 mM TBP or 1% (w/v) DTT, 40 mM Tris, 0.2% (w/v) Bio-Lyte (ampholyte), pH 3–10 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 2 mM TBP or 1% (w/v) DTT, 40 mM Tris, 0.2% (w/v) Bio-Lyte (ampholyte), pH 3–10 (2)Allow sufficient time for full denaturation and solubilization. It is often beneficial to allow the sample to sit at room temperature in the solubilization solution for one hour before applying to IEF. (3)Remove insoluble protein complexes by centrifugation (>10,000 x g) prior to isoelectric focusing.
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