Dear Benjamin Onyebuchi,

Attached please find a Word file containing the procedure to make PNP standard curve.

Hoping this will be helpful,

Rafik

Dear Benjamin Onyebuchi,

Attached please find a Word file containing the procedure to make PNP standard curve.

Hoping this will be helpful,

Rafik

Please find the attached procedure 

I use pNP for cellobiase assay and I prefer it to be in terms of molarity instead of mg/ml which makes enzyme assay calculations easy.

p-nitrophenol (MW = 139.11)

> 10mM pNP stock solution is prepared by dissolving 0.0139 g of pNP in 10 ml of desired buffer.

> Working stock  solution of 1mM pNP is used for preparing std. curve.

> A pNP concentration range of 0 to 100µM (or 0 to 0.1 mM) is used by making appropriate dilutions from the working stock (preferably in 1ml total volume). This range gives a nice linearity and a good regression.

> Absorbance at 420nm is read after addition of 1ml, 1M Na2CO3 to all the samples in the std range including blank.

Good luck.

The following steps we are following  for preperation of p-nitrophenol standard curve while measuring phosphatase activity.

1. Dilute 1 mL of p-nitrophenol solution to 100 mL, it contains 10 ppm p-nitrophenol solution.

2. Pipette out 0,1,2,3,4, and 5 mL of 10 ppm p-nitrophenol solution in 50 mL volumetric flask and add 5 mL distilled water.

3. Add i mL of 0.5 M calcium chloride and 4 mL of 0.5 M NaOH and swarl the flasks.

4. Filter both the sets quickly through whatman no. 42 filter paper. Measure the yellow colour intensity with a blue filter at 420 wavelength.

1. To Dr Rafik Karaman:

the reaction of phosphatase hydrolysis of pNPP is a wrong one, becasue in the reaction is produced a non-coloured p-nitrophenol, but in the nex irreversibel step, after addition of NaOH you obtain a yellow p-nitrophenolate

2. To Dr Amon P Urs

in your procedure you use a distilled water, but in enzymatic assays you MUST use buffer solutions.

3. My propcedure for acid phosphatase (prostatic one, PAP) assay, without any calibration curve,  was:

"Discontinuous acid phosphatase assay using p-nitrophenyl phosphate (p-NPP) as hydrolysis substrate

Catalytic activity of acid phosphatase in 0.1 M acetate buffer at pH 5.0 with p-nitrophenyl phosphate as hydrolysis substrate was determined in discontinuous way. The acidic reaction mixture was alkalized after the course of the reaction. The concentration of the formed p-nitrophenolate ion was measured spectrophotometrically at 400 nm (ε = 18 300 M-1cm-1) [R.L. Van Etten, J.J. McTigue, pH dependence and solvent isotope effects in the hydrolysis of phosphomonoesters by human prostatic acid phosphatase. Biochim. Biophys. Acta 484 (1977) 386-397].

The reaction was started by adding 0.01 ml of enzyme solution (in 10 mM Tris-HCl buffer, pH 7.4, with 100 mM NaCl)  into 0.19 ml substrate solution in 0.1 M acetate buffer, pH 5.0. The reaction was carried out at 200 C for 15 s and was stopped by the addition of 0.8 ml of 0.1 M NaOH solution. The measurements of the p-nitrophenolate absorbance were performed at 400 nm using a double beam UV-VIS Cary 300 spectrophotometer. The enzyme stock solution was kept on ice during the experiment. In a blank sample, the enzyme was not added."

f you measure a coloured substance and you know the molar absorption coefficient ε, you do not need to prepare a calibration curve any more, but you use the Lambert Beer law: A = ε c l and calculate c from c = A / ε  l.

A - absorbance of the sample, ε = molar absorptoion coefficient in M-1 cm-1 (ε = 18 300 M-1cm-1 ), c is molar concentration in M=mol/dm3, l = 1 cm (mostly).

If  you even want to prepare the calibration curve, please do it, but from each experimentasl point calculate ε, and next calculate your mean molar coefficient and use it or use the Excel or other program.

Just for the record, there is a good chapter in the book Pseudomonas Methods and Protocols:

https://link.springer.com/protocol/10.1007%2F978-1-4939-0473-0_12