蛋白质样本制备的原则精简
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蛋白质样本制备的原则精简
蛋白质样本制备的原则
精简
蛋白质样本制备的原则精简
看你要干什么用啊……
Purification纯化
实验方法如Chromatography (Ion exchange,gel filtration,etc.)
Molecular weight and subunit structure 测试分子量并找到有多少subunit
实验方法如 SDS-PAGE:This will allow us to figure out if a single Edman degradation will do or if we will need partial digestions.或者HPLC
Reduction and modification of S-S and –SH groups还原双硫键,改变SH group(前提是你不要这个蛋白的三级结构)
Because S-S and –SH interfere with sequencing procedure and enzymatic/chemical cleavage:disulfide bridges may be holding the protein in a conformation in which the peptide bonds are not reachable by the peptidase.
方法：(1) Reduce disulfide bonds More
Mild:β-mercaptoethanol,HS-CH2-CH2-OH
Strong:DTT
Block –SH
iodoacetic acid,iodoacetate ,I-CH2-COOH
(2) Oxidation:
Performic acid
Separation of subunits or polypeptide chain (by HPLC) if protein is multi-meric