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分子克隆-蛋白表达实验指南（十七）

2020.7.27

Buffers for His purification in denatured conditions:
      配制时加终体积50％的水，之后定容至所需体积。先配pH 8.0的buffer(调pH需要较多NaOH)，再统一配bufferC，D，E，分别调pH
   Buffer B (200ml):
100 mM NaH2PO4        3.12 g NaH2PO4·2H2O (MW 156 g/mol)
10 mM Tris·Cl           0.24g Tris base (MW 121.1 g/mol)
8 M urea                96.1 g (MW 60.06 g/mol)
Adjust pH to 8.0 using NaOH.
          Buffer C (200ml):
100 mM NaH2PO4        3.12 g NaH2PO4·2H2O (MW 156 g/mol)
10 mM Tris·Cl           0.24g Tris base (MW 121.1 g/mol)
8 M urea                96.1 g (MW 60.06 g/mol)
Adjust pH to 6.3 using HCl.
Buffer D (200ml):
100 mM NaH2PO4        3.12 g NaH2PO4·2H2O (MW 156 g/mol)
10 mM Tris·Cl           0.24g Tris base (MW 121.1 g/mol)
8 M urea                96.1 g (MW 60.06 g/mol)
Adjust pH to 5.9 using HCl.
          Buffer E (200ml):
100 mM NaH2PO4        3.12 g NaH2PO4·2H2O (MW 156 g/mol)
10 mM Tris·Cl           0.24g Tris base (MW 121.1 g/mol)
8 M urea                96.1 g (MW 60.06 g/mol)
Adjust pH to 4.5 using HCl.
        Note: Due to the dissociation of urea, the pH of Buffers B, C, D, and E
should be
adjusted immediately prior to use. Do not autoclave.

    SDS-PAGE胶配方
        SDS-PAGE分离胶配方
         5ml8.5ml10ml15ml17.5ml20ml25ml30ml40ml50ml
        6％胶
        水2.64.425.37.99.110.613.215.921.226.5
        30％聚丙烯酰胺混合液11.7233.5456810
        1.5M Tris(pH=8.8)1.32.212.53.84.5556.37.51012.5
        10% SDS0.050.0850.10.150.1750.20.250.30.40.5
        10% AP0.050.0850.10.150.1750.20.250.30.40.5
        TEMED0.0040.00680.0080.0120.0140.0160.020.0240.0320.04

        8％胶
        水2.33.914.66.98.059.311.513.918.523.2
        30％聚丙烯酰胺混合液1.32.212.744.555.36.7810.713.3
        1.5M Tris(pH=8.8)1.32.212.53.84.5556.37.51012.5
        10% SDS0.050.0850.10.150.1750.20.250.30.40.5
        10% AP0.050.0850.10.150.1750.20.250.30.40.5
        TEMED0.0030.00510.0060.0090.01050.0120.0150.0180.0240.03

        10％胶
        水1.93.2345.96.657.99.911.915.919.8
        30％聚丙烯酰胺混合液1.72.893.355.956.78.31013.316.7
        1.5M Tris(pH=8.8)1.32.212.53.84.5556.37.51012.5
        10% SDS0.050.0850.10.150.1750.20.250.30.40.5
        10% AP0.050.0850.10.150.1750.20.250.30.40.5
        TEMED0.0020.00340.0040.0060.0070.0080.010.0120.0160.02

        12％胶
        水1.62.723.34.95.66.68.29.913.216.5
        30％聚丙烯酰胺混合液23.4467810121620
        1.5M Tris(pH=8.8)1.32.212.53.84.5556.37.51012.5
        10% SDS0.050.0850.10.150.1750.20.250.30.40.5
        10% AP0.050.0850.10.150.1750.20.250.30.40.5
        TEMED0.0020.00340.0040.0060.0070.0080.010.0120.0160.02