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Separation of Haemoglobin from Bovine Serum Albumin using the SDS-PAGE and Ion exchange chromatography techniques

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  1. Introduction
  2. Objectives
  3. Results
  4. Discussion
  5. Conclusion

Proteins are large molecules with polar and ionizing groups on their surface. Usually the pH affects the polar charges on the molecule, which causes the proteins to move to its oppositely charged electrode or remain stationary, depending on the buffer chosen. Many different electrophoretic systems have been used in the effective separation of protein mixtures. Two of these techniques are the SDS-PAGE technique which stands for Sodium Dodecyl Sulfate ? Poly Acrylamide Gel Electrophoresis technique and the Ion-Exchange Chromatography (IEC) technique.

The SDS-PAGE technique is often used to determine the molecular weight of a protein. One of the components used in this technique is the chemical substance SDS which stands for Sodium Dodecyl Sulfate. The substance SDS is used in this technique due to some useful characteristics because of its chemical structure. For instance, the hydrophobic tail of dodecyl sulfate interacts strongly with polypeptide chains and it also has two negative charges which collectively overwhelm or swamp any intrinsic charge of the protein. Moreover, SDS also acts as a detergent that disrupts the folding of the secondary and tertiary structures of proteins. These useful characteristics therefore help in finding the relative sizes of the protein molecules used in the electrophoresis. So the number of SDS molecules bound by a polypeptide is proportional to the number of amino acid residues of the polypeptide. Also the electrophoretic mobility of proteins or the rate at which the proteins migrate towards an electrode is inversely proportional to the logarithm of the proteins molecular weight (Garrett & Grisham, 2005, p.150). The other component of this technique is the electrophoresis gel itself. The type of gel used in this technique is the polyacrylamide gel, which is made by polymerizing acrylamide and a cross-linking agent such as N, N'-methylene-bis-acrylamide along with a catalyst. In these gels the protein components are separated according to both the charge and the size of the protein molecules as they move through the pores within the polyacrylamide gel.

[...] Looking at figure it can be seen that the peaks of maximum elution occurred at 5 and 9 mL elution and the greatest elution activity was from 1 to 5mL elution in the elution profile of BSA being eluted from the DEAE column. Again in this elution profile also one of the peaks might have been caused due to contaminants, isomers or coomassie blue as explained above. So in order to identify the isoelectric point, the combinations of 4mL and 5mL elution or the 7mL and 9mL elution should be looked into when taking into consideration a combined observation of the peaks of the CM and DEAE elution profiles. [...]


[...] Total amount of Hb recovered = 4.953 mg Initial amount of Hb present = 6.737 mg x 10 = 67.37 mg % recovery of Hb form CM column = recovered amount/original amount x 100 = ( 4.953 / 67.37 ) 100 = Figure Graph of the mass of haemoglobin in fractions 11-20 in mg from the CM cellulose column against the number of the test tube 11-20. Session 3 Determination of BSA Table The absorbance readings at 595 nm and the concentration for the BSA standard solutions eluted from the CM cellulose column. [...]


[...] So this means that the serum albumin or BSA has an isoelectric point which is in the acidic region of the pH scale and hence it has a lower isoelectric point than hemoglobin ( 6.7 Furthermore it was also noticed that there was a sharp drop in the elution of material as shown in both the elution profiles and this is because the serum albumin has a positive charge below 6.8 and negative charge above 6.8 and therefore above isoelectric point the protein is negative and will bind strongly to anion exchangers but below the isoelectric point, the protein is positive and will bind strongly to cation exchangers. Commenting further on percentage recovery, it is understood from the above that the cation exchangers like the CM column separate hemoglobin and BSA better than the anion exchangers such as the DEAE column. This may not be the case with other proteins which may show different percentage recoveries with the two columns used here. [...]


[...] Raaman, N 2006, Phytochemical techniques, New India publishing Agency, New Delhi. [...]


[...] For the ion exchange chromatography technique, both the CM column and the DEAE column were used to separate the proteins, hemoglobin and serum albumin. Firstly, looking at the results of the technique on hemoglobin, as shown under the results section, session 1 and we can see that hemoglobin is slightly well separated in the cation exchanging, CM column than in the anion exchanging, DEAE column. The results of the percentage recovery calculations prove this, as the percentage recovery of hemoglobin in CM column was higher than for DEAE column. [...]

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