Electrophoresis Separation Of Proteins Cytochrome C, Myoglobin, Hemoglobin, And serum albumin By Using Isoelectric Focusing System (Ief) Electrophoresis Separation of Proteins Cytochrome C, Myoglobin, Hemoglobin, and Serum Albumin by Using Isoelectric Focusing System (IEF) Introduction Proteins are composed of amino acids. All amino acids are amphoteric molecules consisting of three types of amino acids: neutral, acidic, and basic. Thus, for any protein there is a characteristic pH, called the isoelectric point (pI), at which the protein has no net charge and therefore will not move in the electric field. Electrophoresis takes advantage of this characteristic.
Proteins are electrophoreased, and the most negatively charged protein moves closest to the cathode, and the most positively charged protein moves closest to the anode. Cytochrome C was expected to move closest to the cathode, and serum albumin was expected to move closest to the anode. Only cytochrome C was expected to move to the cathode. The other three proteins were expected to move toward anode. The purpose of electrophoresis was to see how a difference in pI makes a difference in the electrophoretic mobility of protein. Materials and Methods Four proteins were electrophoreased by using the Tris-Glysin buffer of pH 8.
6 and a horizontal agarose gel 1. 1 % in isoelectric focusing (IEF) at a voltage of 175 V and at a current of 79 mA. The agarose gel was made by mixing 0. 18 g of agarose in 1.
The Essay on Gel Electrophoresis Adventure
Intro The final goal of this lab was to successfully measure the size of different samples of DNA by placing each sample into a well in agarose gel and running a current through a charged chamber. The DNA samples will move through the gel towards the positive charge. Ideally, the DNA will move and create and sequence of smallest to largest. This lab exposes us to DNA technology. Backround Gel ...
5 ml of Tris-Glysin buffer with a pH of 8. 6. That is 100 % 0. 18 / (0. 18 + 15) = 1. 1% of agarose gel.
15 l of each protein sample was loaded into each sample application well on the agarose gel without mixing with glycerol solution. After the agarose gels were placed on the stage of the electrophoresis chamber, Tris-Glysin buffer of pH 8. 6 was filled in the electrophoresis chamber carefully until the agarose gels were slightly covered with the buffer. Four protein had electrophoreased for about 50 minutes. The agarose gels were removed from the electrophoresis chamber and stained overnight with the Coomassee Blue to visualize proteins in the agarose gel. Results Well 1 Cytochrome C pI 10.
2 Well 2 Myoglobin pI 7. 2 Well 3 Hemoglobin pI 6. 8 Well 4 Serum albumin pI 4. 8 Sample Volume 15 l PH of buffer 8. 6 Voltage 175 V Current 79 mA Running Time 0. 8 hr Table 1 shows the conditions of this IEF electrophoresis.
15 l of each cytochrome C, myoglobin, hemoglobin, and serum albumin were loaded into the well as indicated in Table 1. Well 1 is the bottom well in Figure 1. A voltage of 175 V and a current 79 mA was applied in the buffer of pH 8. 6 for 50 minutes, and bubbles were observed on the electrode during the electrophoresis.
Figure 1 shows that cytochrome ( 1 in Figure 1) moved closest to the cathode, and serum albumin ( 4) moved closest to the anode. Myoglobin ( 2) and hemoglobin ( 3) moved toward the anode, but hemoglobin moved farther from the well than myoglobin. Discussion The results support the original hypothesis that cytochrome C will move closest to the cathode, and myoglobin and hemoglobin will move to the anode with serum albumin being the closest to the anode. These results clearly show the relationship between movement of proteins and their isoelectric point (pI).
The greater the difference is between pI of proteins and pH of the buffer, the farther the proteins are from the well in this experiment. The protein with a higher pI than the pH of the buffer was positively charged because it accepted hydrogen ions from the buffer.
Then this positively charged protein moved to the negative region, or cathode because it was attracted by hydroxyl ions formed at the cathode by the electrode reactions. When this protein bonded with hydroxyl ions, it became neutral and stopped its movement. On the other hand, the protein with lower pI than the pH of the buffer was attracted by the positive region, or anode, where hydrogen ions were formed. Since this protein released hydrogen ions to the buffer, it became negatively charged and moved to the anode to bond with hydrogen ions to become neutral. The IEF electrophoresis using agarose gels have been used by researchers, and this technique has proved to be an efficient method for separating small quantities of proteins.
The Essay on Estimation of protein concentration
Protein assays are designed to measure the total protein in a solution. Protein assays are quantitative if the protein to be assayed is available in sufficient quantity such that one is able to use it to create a standard curve. If this cannot be achieved, then a standard protein, such as albumin, may be used for a standard curve with the understanding that the results on the unknown protein are ...
U. Ravnskov (1975) states in his article Low molecular weight proteinuria in association with paroxysmal myoglobinuria that agarose gel electrophoresis is a great method to separate myoglobin and hemoglobin. The difference between hemoglobin and myoglobin in pI is 0. 4, yet the IEF horizontal agarose gel electrophoresis with 15 l of quantity visualized this difference. A study performed by C. Caudie, O.
Allauzen, J. Bancel, and R. Later (2000) used agarose gel IEF and IgG immuno revelation to detect IgG oligoclonal bands (OCB).
Their conclusion was that IEF with immune detection is the most sensitive and specific test for the detection of chronic CNS inflammation. Similar research was performed by J.
Lunding, R. Midgard, and CA. Vedeler (2000) who compared the superiority of IEF, agarose gel electrophoresis (AGE), and IgG index in sensitiveness and specificity in detecting nervous system disorder. Restricted OCB were found in IEF and AGE, and the researchers found that more accurate results were obtained from IEF. Also, IEF was far better than IgG index in determining intrathecal IgG synthesis. As researchers recommended IEF, the migration of all four proteins were successful with IEF using the horizontal agarose gel even with the small amount of protein samples.
This technique could be used in analysis, purification, and detection of proteins. Improvements could be made in the resolution of the protein band in the agarose gel and experimental time. Improvement in resolution could be achieved by reducing the diffusion. An increase of the viscosity of the agarose gel reduces the diffusion, and resolution would therefore increase. The adverse effect of this method is that it would slow down the experiment because the increase of viscosity of the agarose gel increases the friction of proteins. Increasing the experimental time reduces the resolution and thus is not always a successful method to improve resolution.
The Review on The Effects Of Crude Protein On Antler Growth In White Tailed Deer
The Effects of Crude Protein on Antler Growth in White Tailed Deer Introduction White tailed deer go through drastic endocrine and physiological changes during the year. Due to this reason, their requirements for nutritional supplements are changing quickly. However, the requirement for crude protein seems to be very important for antler growth and to allow optimal development. The present study ...
This method would not be a good method for the proteins cytochrome C, myoglobin, hemoglobin, and serum albumin because Figures 2, 3, 4, and 5 show that hemoglobin and serum albumin are greater in size. That is, hemoglobin and serum albumin tend to be influenced by the friction. Another method to improve the resolution is to increase the strength of the electric field. This method also reduces the time of the migration of the proteins. The only thing to be careful with about this method is the temperature of the agarose gel because the high electric field produces heat, and this might cause the proteins to be denatured. Literature Cited Ravnskov, U.
(1975, February).
Low molecular weight proteinuria in association with paroxysmal myoglobinuria. [Abstract] Clin Nephrol 1975 Feb; 3 (2).
65-9.
Retrieved January 31, 2001 from the WWW: web Caudie, C. , Allauzen, O. , Bancel, J. , and Later, R.
(2000 March-April).
Role of isoelectric focusing of cerebrospinal fluid immunoglobulin G in the early biological assessment of multiple sclerosis. [Abstract] Annales de Biologie Clinique. Vol. 58, Issue 2, 187-93. Retrieved February 2, 2001 from the WWW: web Lunding, J.
, Midgard, R. , and Vedeler, CA. (2000 Nov).
Oligoclonal bands in cerebrospinal fluid: a comparative study of isoelectric focusing, agarose gel electrophoresis and IgG index. [Abstract] Acta Neurol Scand 2000 Nov; 102 (5).
322-5.
Retrieved February 2, 2001 from the WWW: web Natural Toxins Research Center at Texas A&M University. Isoelectric focusing. Retrieved January 29, 2001 from the WWW: web Bibliography Literature Cited Ravnskov, U. (1975, February).
Low molecular weight proteinuria in association with paroxysmal myoglobinuria. [Abstract] Clin Nephrol 1975 Feb; 3 (2).
65-9. Retrieved January 31, 2001 from the WWW: web Caudie, C. , Allauzen, O. , Bancel, J.
, and Later, R. (2000 March-April).
The Term Paper on Web Services part 1
Web Services. Web Services are not implemented in a monolithic way, but rather represent a collection of several related technologies. A new language was developed in the Internet back in 1999. What were the reasons for that? The old one was called HTML, and it was a gear for the whole internet, especially for the area called World Wide Web, development process. Now the Web outgrew its creator and ...
Role of isoelectric focusing of cerebrospinal fluid immunoglobulin G in the early biological assessment of multiple sclerosis. [Abstract] Annales de Biologie Clinique.
Vol. 58, Issue 2, 187-93. Retrieved February 2, 2001 from the WWW: web Lunding, J. , Midgard, R. , and Vedeler, CA. (2000 Nov).
Oligoclonal bands in cerebrospinal fluid: a comparative study of isoelectric focusing, agarose gel electrophoresis and IgG index. [Abstract] Acta Neurol Scand 2000 Nov; 102 (5).
322-5. Retrieved February 2, 2001 from the WWW: web Natural Toxins Research Center at Texas A&M University.
Isoelectric focusing. Retrieved January 29, 2001 from the WWW: web >.
