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Isolation & Characterisation of Proteins. The purpose of conducting this experiment is to study protein separation using two different methods, which are the SDS-polyacrylamide gel and ion exchange chromatography

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Introduction

Name: Ng Yen Pheng Student ID: 22353046 Day and date: Tuesday, 3 April 2012 Title: Isolation & Characterisation of Proteins Aim: The purpose of conducting this experiment is to study protein separation using two different methods, which are the SDS-polyacrylamide gel and ion exchange chromatography. This practical also aims to study what types of ion exchangers which are more suitable to separate different types of proteins. This experiment also aims to compare the separation of proteins by SDS-PAGE and ion exchange chromatography. Results: Part A: SDS-PAGE Figure 1: Image of SDS-Polyacrylamide gel electrophoresis. Table 1: Distance migrated (cm) by different length of protein fragments denatured by SDS Bands Molecular Weight, MW (kDa) Log10 (MW) Distance migrated (mm) 1 250 2.40 14 2 130 2.11 24 3 100 2.00 33 4 70 1.85 43 5 55 1.74 48 6 35 1.54 - 7 25 1.40 66 8 15 1.18 75 9 10 1.00 87 [( - ) in the table indicates that the band is missing] Figure 2: Graph of log (Molecular weight) against distance migrated for marker. Table 2: Distance migrated and size of fragments of 3% haemoglobin being denatured by SDS with different dilution factor in the presence of 1% of BSA. Dilution factor Distance migrated (mm) Log (Molecular weight) Molecular Weight (kDa) 1/5 (Sample A) 43 1.819 65.892 90 0.929 8.497 1/10 (Sample B) 44 1.780 63.083 91 0.910 8.135 1/50 (Sample C) 45 1.781 60.392 92 0.891 7.788 Calculation: To calculate molecular weight for samples Using the equation in the graph above, y = -52.836 x + 139.1 where x = log (MW) For sample A, when the protein fragment has migrated 43 mm y = 43 43 = x = = 1.819 Molecular weight = 101.819 = 65.892 Another band in sample A migrated 90 mm y = 90 90 = -52.836 x + 139.1 x = = 0.929 Molecular weight = 100.929 = 8.497 For sample B, when the protein fragment has migrated 44mm y = 44 44 ...read more.

Middle

of DEAE column, y = 0.0053x in which y = absorbance and x = albumin mass By using equation: y = 0.0053x Mass of serum albumin in eluent fraction in tube 1, �g 0.069 = 0.0053x x = = 13.02 �g/0.1ml When 0.1ml consists of 13.02 �g of serum albumin 1ml consists of = 130.19 �g Concentration of serum albumin in each eluent fraction in tube 11, g/ 100mL When the mass of BSA present is calculated to be 130.19 �g in 1ml solution Thus, concentration is = 130.19 �g/ml Same method of calculation was applied to tube 1 - 10 and included diluted sample in order to calculate mass and concentration of serum albumin in eluent fraction. Total mass of serum albumin recovered in CM column in tube 1 - 2, �g = 130.19 + 145.28 + 226.41 + 271.70 + 292.45 + 264.15 + 256.60 + 305.66 + 271.70 + 283.02 = 2447.16 �g Percentage recovery of BSA in CM column, % = [ ] � 100% = x 100% = 37.27 % Figure 8: Elution profile of mass of serum albumin against test tube in CM column. Discussion: Part A: SDS-PAGE When proteins are to be separated using electrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is often chosen. SDS is a strong detergent which can denature proteins, unravelling the tertiary structure of proteins to secondary or primary (Puri, 2006). This is due to the interference to the hydrophobic interactions of proteins which act to stabilize them (Puri, 2006). SDS molecules have negative charges which can mask the proteins intrinsic charges (Puri, 2006). In SDS-PAGE, proteins denatured by SDS require different time. The 5% stacking region in the SDS-PAGE gives proteins time to be completely denatured, ensuring that the samples in all the columns have been denatured and start running in the gel at the same time. From the gel obtained in figure 1, 8 bands were found instead of 9. ...read more.

Conclusion

Of the two methods to separate proteins, IEC would yield a more accurate result as compared to SDS-PAGE. SDS-PAGE only shows bands which, for the determination of types of proteins present, it has to be referred to a protein ladder. Sometimes the bands may not be obvious enough to determine the distance migrated. Hence determination of types of proteins may not be accurate. In IEC, proteins bound to the charged resin beads to different extent according to their polarity. The results then passed to the detector to be analysed, presenting types of proteins present as peaks at particular retention time. Peaks and retention time are different for different proteins. Hence this will produce result which is much more accurate. Besides, proteins sizes may vary greatly. In electrophoresis, pore size is even throughout the gel. Proteins with smaller size may not be well separated in SDS-PAGE, whereas in IEC, proteins will be adsorbed to the resin beads to different extent, according to their polarity. Proteins which are not adsorbed onto the resin beads will be flowed out first then only the bound proteins will be "knocked off" from the resin beads. This enables better separation of proteins. Conclusion: In SDS-PAGE, the greater the concentration of sample, the slower the sample will be travelling at. Molecular weight of haemoglobin for 3 dilution factor of 5, 10 and 50 are 8.497kDa, 8.135kDa and 7.788kDa. While molecular weight for BSA in 3 different dilution factor is 65.892kDa, 63.083kDa and 60.392kDa. This indicated that high molecular weight BSA travelled slower than haemoglobin. Haemoglobin which present at 3% travels slower that BSA which present at only 1%. DEAE column is the most suitable method used to measuring haemoglobin as it has a high percentage of recovery which is 98.28% while CM column only 68.48%. However, CM column separates BSA better since it has higher percentage of recovery which is 37.27% while DEAE only 15.84%. IEC separates proteins better than SDS-PAGE does. ...read more.

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