Acid phosphatase - week 2

Protein and enzyme assay

Quantification of proteins in various aliquots/fractions and phosphatase activity assay will be carried out during this week. Protein assay will require only about 1 hour while enzyme assay may require 2-3 h.
All the assays must be carried out in duplicate.

1. Protein assay:

The method you will be using for quantitation of protein is that developed by Bradford (1976). This is a simple but sensitive method and can be used to determine amounts as low as 1-20 µg (microassay procedure) or 20-140 µg (standard assay procedure).
For simplicity, you should carry out the micro assay for all fractions. This will require that you dilute your fractions accordingly, since the assay is accurate only up to 1.0 absorbance units.

You have to prepare a standard curve using known amounts of proteins. You are provided with Bovine gamma globulin at a concentration of 1.56 mg/ml of water. This is your stock solution.

Microassay procedure

1. Use the 10X diluted substock (0.156 mg/ml) standard curve.
Prepare a working concentration of 1/10 dilution of stock by adding 0.1 ml of stock and making up to 1 ml with dist. water. Vortex and label as standard substock.

2. Pipette the volumes of substock and dist. water given in the Table 1 into clean test tubes. Prepare two blank controls (800 µl water only). Also pipette 800 µl of your HOM, P1,...., SN4 isolate (Table 2) and every other fraction from gel filtration.

3. Add 200 µl of concentrated dye (caution: corrosive!) using pipetman P200. Due to the high viscosity of the solution, suck up the concentrate slowly.

4. Incubate at room temperature for 5 min or more (but no more than 1 h).

5. Measure the absorbance at 595 nm in the Spectronic 21 spectrophotometer. Use plastic cuvettes. Start with the blank samples, then move up to higher concentrations of your standard. Re-use the cuvettes for all your samples.

6. Draw a standard curve and determine the amount of protein contained in each sample. A typical standard curve is shown here.


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