실험실 실무 사용자를 위한 상세한 웨스턴블로팅법 정리

소개

실험실에서 단백질발현측정에 흔히 쓰여지는 복잡하고도 값비싼 웨스턴블롯기법을 손쉽게 볼 수 있도록 정리하였습니다.

목차

*Western blot 정리.
단백질 -1차Antibody-2차 Antibody-발색체

본문내용

1*Western blot 정리.
<Northern Blot> DNA-DNA hybridization
<Southern Blot> RNA-DNA
<Western Blot> Protein-Protein
원리:-항체를 detection tool로 이용.

 
 
단백질 -1차Antibody-2차 Antibody-발색체
 
1) 시료 전처리- Western sample 만들기.
protein lysis (단백 동화)
↓
quantification (정량)
↓
boiling(sample +buffer+4x sample buffer)⇒양 맞춤. gel loading 조건을 만들어줌.
95℃에서 9~10분간 boiling함.
- 자세한 Sample Quantification for Biochemistry-
Homogenization
1. Weigh the Tissue and make equally average weight. (10~20mg)
2. Add PBS which volume is 30 times amount of Tissue weight. and add 1% protease inhibitor. ex) if hippo 10mg → 300 μl PBS
3. homogenize
4. 12000rpm 4℃, 20min centrifuge.
5. move supernatant another tube →keep in deep freezer.
Quantification
1. Preparing working solution - by using Quant-it™ Protein Assay kit
-each sample need total 190μl working solution.
-standard 1,2,3 also need 190μl working solution.
-because of bubble it`s better make a little more amount.
protein reagent : protein buffer = 1: 200

참고자료

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