The established protocol for ensilication requires 50 mM of Tris buffer at pH 7.0 with no sodium present. Therefore, 3.03 g of Trizma-Base (MW: 121.14 g/mol, cat: T666-1kg, Sigma UK) was weighed out to make 500 ml buffer. In a beaker, 400 ml of ultrapure (MilliQ, MilliPore UK) water was added to the weighed Trizma and using a magnetic stirrer this was dissolved. The pH was set using 32% (v/v) hydrochloric acid. Finally, the solution was filled up to 500 ml final volume with ultrapure water.
Recombinant expression in E. coli
Bacterial medium, Luria Broth (LB) contains 10 g/L Tryptone, 5 g/L Yeast Extract, 5 g/L Sodium Chloride, Sigma, in ddH2O. Autoclave after preparation and use when needed.
Agar plates were made of LB, 10 g/L Tryptone, 5 g/L Yeast Extract, 10 g/L Sodium Chloride, with 15 g/L added agarose, Sigma, in ddH2O. Autoclave after preparation and pour plates when hand warm in a laminar flow-hood.
Bacterial stocks were made by addition of 30% glycerol and stored at -80 °C until further use.
Buffers for HisTag purification:
|IMAC25 (binding)||0.5 M||0.1 M||25 mM||pH 8.0 in ddH2O.|
|IMAC500 (elution)||0.5 M||0.1 M||500 mM||pH 8.0 in ddH2O.|
pH solution with HCl. Autoclave or filter 0.45 μm for use in the FPLC.
ELISA 5’-5’ tetra-methyl-benzidine (TMB, Sigma, UK) substrate
|stock||10 μg / ml||0.1 M pH 6.0||30 % (v/v)|| TMB in DMSO or EtOH |
NaAct stock 1M pH with citric acid.
|in 10 ml||100 µL||10 ml||1.5 µL|
5’-5’ tetra-methyl-benzidine (TMB, Sigma, UK) was dissolved in 1 ml of dimethylsulfoxide (DMSO, Sigma, UK) or EtOH at 10 μg / ml and 100 μL was added to 10 ml of 0.1M sodium acetate pH 6.0 (with citric acid) with addition of 1.5 μL of 30% (v/v) H2O2.
Sodium Phosphate Buffers
|pH||Volume (mL) of 1 M Na2HPO4||Volume (mL) of 1 M NaH2PO4|