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.
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.
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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