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 3.1Ultraviolet absorbance of protein

Absorbance, A of samples

wavelength/nm Blank (water) 1mg/ml BSA #54 #159250 0 0.511 0.252 0.995

260 0 0.575 0.294 1.090

270 0 0.783 0.423 1.048

280 0 0.876 0.494 0.889

290 0 0.510 0.293 0.464

300 0 0.111 0.048 0.099

310 0 0.049 0.010 0.030

320 0 0.039 0.004 0.022

330 0 0.036 0.003 0.020

340 0 0.033 0.001 0.018

350 0 0.034 -0.001 0.014

360 0 0.033 -0.001 0.014

Table 1: Absorption Spectra over different wavelength (nm)

250 260 270 280 290 300 310 320 330 340 350 360-0.2

0

0.2

0.4

0.6

0.8

1

1.2

Absorbance, A of samples

Blank (water)1mg/ml BSA#54#159

wavelength/nm

Absorbance

3.1.3 Questions

1. Using E1% 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 E0.1%. We have to use E0.1%, instead of E1%, because of the initial dilution of BSA.

For Unknown 159,

Absorbance at 280nm wavelength = 0.889

Using E1%=10.0, E0.1% = 1.00

Protein content of Unknown 159 =0.8891.00

1.0mg/ml

= 0.889 mg/ml (3 significant figures)

Using E1%=6.67, E0.1% = 0.667

Protein content of Unknown 159 =0.8890.667

1.0mg/ml

= 1.33 mg/ml (3 significant figures)

For Unknown 54,

Absorbance at 280nm wavelength = 0.494

Using E0.1%=1.00,

Protein content of Unknown 54 =0.4941.00

1.0mg/ml

= 0.494 mg/ml (3 significant figures)

Using E0.1%=0.667,

Protein content of Unknown 54 =0.4940.667

1.0mg/ml

= 0.741 mg/ml (3 significant figures)

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 tryptophan. 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.