LSM 1101- Lab Report

(Practical 2: Quantitative Protein Estimation)

Group 1- Bench 6 Date of Experiment: 28th Aug 2009 Members name: Kang Bo Han Abraham (U0900023J) Karthig s/o (U0804997)

Experiment 3.1 Ultraviolet absorbance of protein Absorbance, A of samples Blank (water) 1mg/ml BSA 0 0.511 0 0.575 0 0.783 0 0.876 0 0.510 0 0.111 0 0.049 0 0.039 0 0.036 0 0.033 0 0.034 0 0.033

wavelength/nm 250 260 270 280 290 300 310 320 330 340 350 360

#54 0.252 0.294 0.423 0.494 0.293 0.048 0.010 0.004 0.003 0.001 -0.001 -0.001

#159 0.995 1.090 1.048 0.889 0.464 0.099 0.030 0.022 0.020 0.018 0.014 0.014

Table 1: Absorption Spectra over different wavelength (nm)

Absorbance, A of samples
1.2
1

0.8

Absorbance 0.4

1 g/ l BS

#54 #159

0.2 0
-0.2 250 260 270 280 290 300 310 320 330 340 350 360 wavelength/nm

0.6

Blank (water)

3.1.3 Questions
1. Using E 1% value of 10 representing the absorption of a protein concentration of 10mg/ml, the absorption of the same protein concentration of 1mg/ml will be E 0.1%. We have to use E0.1%, instead of E 1%, because of the initial dilution of BSA. For Unknown 159, Absorbance at 280nm wavelength Using E 1%=10.0, E0.1% = = = = Using E 1%=6.67, E0.1% = = = For Unknown 54, Absorbance at 280nm wavelength Using E 0.1%=1.00, Protein content of Unknown 54 = = Using E 0.1%=0.667, Protein content of Unknown 54 = = 1.0mg/ml 0.741 mg/ml (3 significant figures) 1.0mg/ml 0.494 mg/ml (3 significant figures) = 0.494 0.889 1.00 1.0mg/ml 0.889 mg/ml 0.667 1.0mg/ml 1.33 mg/ml (3 significant figures) (3 significant figures)

Protein content of Unknown 159

Protein content of Unknown 159

2.

The Beer-Lambert Law states that the absorption of light by the solutes is directly proportional to the concentration of the solutes. In this case, it will be due to the protein content of the solutions. The 1mg/ml of BSA used shows a maximum absorption of 0.876 at 280nm. Since the BSA sample used is pure and does not contain any contaminants, we can deduce that all the absorption recorded is due to the protein content present in the BSA. In the sample of #159, the maximum absorption recorded is 1.090 at the wavelength of 260nm. Since a pure protein will exhibit a maximum absorption at wavelength 280nm, we can infer that the sample of #159 is most probably contaminated with other solutes. Hence it is probable that it does not contain pure proteins or it might have a lower concentration of tyrosine or trypt ophan. However, as it shows a higher absorption value than that of BSA, we can conclude that this sample of #159 contains a higher concentration of solutes (protein content) than that of 1mg/ml of BSA. For the sample of #54, the maximum absorption recorded is 0.494 at the wavelength of 280nm. This reading indicates that the sample is most likely uncontaminated. On the other hand, due to its lower absorption value, the sample probably has a lower concentration of protein as compared to both BSA and #159.