The Spectrophotometer Determination of Protein Concentrations and the Effects Sodium Dodecyl and Triton X100 Have on Protein Concentration.

2095 Words Sep 27th, 2011 9 Pages
The Spectrophotometer Determination Of Protein Concentrations And The Effects Sodium Dodecyl Sulphate And Triton X-100 Have On Protein Concentration.

Spectroscopy is used as a collective term for all the analytical techniques based on the interaction of light and matter. Spectrophotometry is one of the branches of spectroscopy where we measure the absorption of light by molecules that are in a gas or vapour state or dissolved molecules/ions (Tombs,, 1959). Spectroscopy is the use of electromagnetic radiation by a sample in order to identify components of the sample (qualitative analysis) or to measure the amount of material in that sample (quantitative analysis) (Tombs,, 1959). Absorption is a process in which
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The BIORAD dye was firstly prepared by dissolving 0.225g of Coomassie Brilliant Blue G-250 (Merck, South Africa), in 67.5ml of Methanol (Riedel-de-Haёn, Germany) and 291ml of 85% (w/v) Phosphoric Acid, diluted to 450ml with dH2O. Six test tubes were labelled SC1-6 and the 0.1mg/ml stock of Bovine Serum Albumin (Roche Diagnostics, Germany) and dH2O was pipette into their respective test tubes according to Table 1.

Table 1: Table of the BSA and dH2O volumes to be pipette and placed in the spectrophotometer to obtain absorbencies for the standard curve

Test Tube Number Total Volume (μl) Protein (BSA) Stock (μl) H2O (μl) Amount of Protein (μg)
1 2400 0 2400 0
2 2400 50 2350 5
3 2400 100 2300 10
4 2400 150 2250 15
5 2400 200 2200 20
6 2400 250 2150 25

600μl of the BIORAD dye agent was pipette into each test tube making up the volume to 3000μl. All the test tubes were then covered with Parafilm and placed in the Vortex (Lab Pro cc) for a few seconds and they were incubated at room temperature for 5-60 minutes. Simultaneously another set of test tubes were labelled X1-8 and pipette with 0.1mg/ml Lysozyme stock (Merck, Germany), BSA, 1% (w/v) Sodium Dodecyl Sulphate prepared with dH2O, 1% (v/v)
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