EDVO-Kit # 1 1 1 Electrophoretic Properties of Native Proteins tudy Questions An
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EDVO-Kit # 1 1 1 Electrophoretic Properties of Native Proteins tudy Questions Ansu estions 1. Under the conditions of the electrophoresis (pH 7.8), which of the proteins (BSA, ovalbumin. Cytochrome C. Lysozyme) are net positive? Which are net negative? 2. Based on your observations, would you expect ovalbumin to have more or less lysine and arginine residues than lysozyme? Why? A sample of native protein is submitted to native gel electrophoresis. After the electrophoresis was run for several hours, it was found that the protein did not enter the gel from the sample well [did not migrate). What is the most likely explanation for this observation What experimental condition couid you change that would allow the protein to migrate? 3. Consider the following information about the proteins used in this experiment: 4. ApproximateIsoelectric Molecular Weight Point (pH Protein Cytochrome C Lysozyme Ovalbumin BSA 12.000 14,000 43.000 68.000 10.7 11.2 All these proteins are spherical in shape, therefore, increasing mo- lecular weight corresponds to increasing size. The results of the electrophoresis at pH 7.8 should show that ovalbumin migrates a greater distance towards the positive electrode than BSA. The results shouid also show that Lysozyme migrates a greater distance towards the negative electrode than Cytochrome C. Explain the results using the information provided above. TEK. Duplication of this document in conjunction with use of accompanying reagents, ls permitted for classroom without the written consent of EDVOTEK, Inc. Copyrigh1989,1992.1994,1995,1997,1998, 1999,2000, 2005 EDVOTEK, Inc., al rights reserved EVT 011285AM The Biotechnology Education Company-800-EDVOTEK www.edvotek.comExplanation / Answer
1. BSA, Ovalbumin is net negative charge. When the pH > pI, a protein has a net negative charge
Cytochrome C ], Lysozyme is net positive charge. pH < pI, a protein has a net positive charge.
2. Ovalbumin has less lysine and argine residues. Because it is posituve charged amino acids and its side chain pKR is greater than 10. So, for this type of basic amino acids the isoelectric pH is high due to this argine and lysine residues.
3. Decrease the cross linker in the gel.
Alter the PBS pH
Flipping the voltage some times wrongly connect between anode and cathode.
4. Ovalbumin and BSA are net negative charge and move towards positive electrode. in this Ovalbumin has a less molecular weight 43000 so it migrate faster than BSA has high molecular weight 68000.
Same way cytochrome C and lysozyme has net +ve charge so move toward negative electrode. In this lysozyme has 14000 it not move large distance toward positive electrode so it is near to negative electrode.
the order of separtion from -ve to +ve electrode is,
Lysozyme - Cytochrome C - BSA - Ovalbumin.
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