Useful buffers

Biological buffers

Tris buffer
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.

Hydrolysation of TEOS for ensilication
In a glass beaker, 20 ml of TEOS (cat: 86578-1L, Sigma UK) was added to 20 ml of ultrapure (MilliQ) water with stirring at 350 rpm at room temperature. Hydrochloric acid 32% (v/v) was added to this solution in a 1:500 ratio to initiate the acid catalysis of TEOS into Si(OH)4. Once both components formed a clear homogeneous solution, ethanol was evaporated with slow stirring (60 rpm) for 20 minutes. The solution was then ready to be used within a window of 20 minutes.

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:

NaCl Tris Imidazole
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

stock10 μg / ml0.1 M pH 6.030 % (v/v) TMB in DMSO or EtOH
NaAct stock 1M pH with citric acid.
in 10 ml100 µL10 ml1.5 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.