I would dissolve the substrate in Methanol to get a stock concentration of 50 mg/ml or 184 mM. I will then try 74 fold dilution in your reaction buffer to achieve a final concentration of 2.5 mM. MeOH will also undergo dilution by similar fold.

Have you tried a 50% v/v Methanol or ethanol in water solution? Is it possible for you to use a solvent like ethylene glycol? we routinely use a solvent for preparing substrate stocks.

Hi there,

According to the specification sheet of product N3512 from Sigma, 4-Nitrophenyl α-L-arabinopyranoside is soluble up to 20mg/mL in 50% ethanol (https://www.sigmaaldrich.com/Graphics/COfAInfo/SigmaSAPQM/SPEC/N3/N3512/N3512-BULK________SIGMA____.pdf)

which is more convenient than 100% methanol for an enzyme assay.

An answer in many steps:

1- Avoids dissolution in water

2- To prepare 100 ml of stock solution, put 5g of p-Nitrophenyl-α-L-arabinofuranoside, in 100ml volumetric flask then fill flask with 100% methanol (MeOH).

3- Calculation of stock solution concentration: C = m/ (V*M) A.N: C=5/ (0.1*271.22) = 184.35mM. Molecular Weight: p-Nitrophenyl-α-L-arabinofuranoside is 271.22.

4-Prepare the appropriate the buffer solution.

5- Use buffer solution as diluent, by using a formula for dilution is :

Cf*Vf= Ci*Vi

For example:

Ci=184.35 mM

Cf= 2.5 mM

Vf= 100 ml but we don’t know the value of Vi?

📷Vi= ? Vi=Cf*Vf/Ci AN : Vi=2.5*100/184.35 =1.36 Ml ----Vi=1.36mL

6- In conclusion, to prepare 100 mL of buffer substrate solution, I will measure 1.36 mL from stock solution of substrate (184.35 mM) and put it in a volumetric flask of 100 ml, after this , fill the flask with buffer solution to measure 100ml. Then we will have 100ml of substrate solution 2.5mM.

I would like to say many many thanks to all of you for your kind suggestions. Happy research!