Electrophoresis Separation Of Proteins Cytochrome C Myoglobin free essay sample

Electrophoresis Separation Of Proteins Cytochrome C, Myoglobin, Hemoglobin, And Serum Albumin By Using Isoelectric Focusing System ( Ief ) Essay, Research Paper Electrophoresis Separation of Proteins Cytochrome C, Myoglobin, Hemoglobin, and Serum Albumin by Using Isoelectric Focusing System ( IEF ) Introduction Proteins are composed of aminic acids. All aminic acids are amphiprotic molecules dwelling of three types of amino acids: impersonal, acidic, and basic. Therefore, for any protein there is a characteristic pH, called the isoelectric point ( pi ) , at which the protein has no net charge and hence will non travel 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 travel closest to the cathode, and serum albumen was expected to travel closest to the anode. Merely cytochrome C was expected to travel to the cathode. The other three proteins were expected to travel toward anode. The intent of cataphoresis was to see how a difference in pi makes a difference in the cataphoretic mobility of protein. Materials and Methods Four proteins were electrophoreased by utilizing the Tris-Glysin buffer of pH 8.6 and a horizontal agarose gel 1.1 % in isoelectric focussing ( IEF ) at a electromotive force of 175 V and at a current of 79 ma. The agarose gel was made by blending 0.18g of agarose in 1.5ml of Tris-Glysin buffer with a pH of 8.6. That is 100 % * 0.18 / ( 0.18 + 15 ) = 1.1 % of agarose gel. 15? ? cubic decimeter of each protein sample was loaded into each sample application good on the agarose gel without blending with glycerol solution. After the agarose gels were placed on the phase of the cataphoresis chamber, Tris-Glysin buffer of pH 8.6 was filled in the cataphoresis chamber carefully until the agarose gels were somewhat covered with the buffer. Four proteins had electrophoreased for approximately 50 proceedingss. The agarose gels were removed from the cataphoresis chamber and stained overnight with the Coomassie Blue to visualise proteins in the agarose gel. Consequences Well 1 Cytochrome C pI 10.2 Well 2 Myoglobin pi 7.2 Well 3 Hemoglobin pi 6.8 Well4 Serum albumin pI 4.8 Sample Volume 15? ? cubic decimeter PH of buffer 8.6 Voltage 175 V Current 79 ma Runing Time 0.8 hour Table 1 shows the conditions of this IEF cataphoresis. 15? ? cubic decimeter of each cytochrome C, myoglobin, haemoglobin, and serum albumen were loaded into the well as indicated in Table 1. Well 1 is the bottom well in Figure 1. A electromotive force of 175 V and a current 79 ma was applied in the buffer of pH 8.6 for 50 proceedingss, and bubbles were observed on the electrode during the cataphoresis. Figure 1 shows that cytochrome ( *1 in Figure 1 ) moved closest to the cathode, and serum albumen ( *4 ) moved closest to the anode. Myoglobin ( *2 ) and haemoglobin ( *3 ) moved toward the anode, but hemoglobin moved further from the well than myoglobin. Discussion The consequences support the original hypothesis that cytochrome C will travel closest to the cathode, and myoglobin and haemoglobin will travel to the anode with serum albumen being the closest to the anode. These consequences clearly show the relationship between motion 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 part, 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 impersonal and stopped its motion. On the other manus, the protein with lower pi than the pH of the buffer was attracted by the positive part, or anode, where H ions were formed. Since this protein released H ions to the buffer, it became negatively charged and moved to the anode to bond with H ions to go impersonal. The IEF cataphoresis utilizing agarose gels have been used by research workers, and this technique has proved to be an efficient method for dividing little measures of proteins. U. Ravnskov ( 1975 ) provinces in his article Low molecular weight albuminuria in association with paroxysmal myoglobinuria that agarose gel cataphoresis is a great method to divide myoglobin and haemoglobin. The difference between haemoglobin and myoglobin in pi is 0.4, yet the IEF horizontal agarose gel cataphoresis with 15? ? cubic decimeter of measure visualized this difference. A survey performed by C. Caudie, O. Allauzen, J. Bancel, and R. Later ( 2000 ) used agarose gel IEF and IgG immunorevelation to observe IgG oligoclonal sets ( OCB ) . Their decision was that IEF with immune sensing is the most sensitive and specific trial for the sensing of chronic CNS redness. Similar research was performed by J. Lunding, R. Midgard, and CA. Vedeler ( 2000 ) who compared the high quality of IEF, agarose gel cataphoresis ( AGE ) , and IgG index in sensitivity and specificity in observing nervous system upset. Restricted OCB were found in IEF and AGE, and the research workers found that more accurate consequences were obtained from IEF. Besides, IEF was far better than IgG index in finding intrathecal IgG synthesis. As research workers recommended IEF, the migration of all four proteins were successful with IEF utilizing the horizontal agarose gel even with the little sum of protein samples. This technique could be used in analysis, purification, and sensing of proteins . Improvements could be made in the declaration of the protein set in the agarose gel and experimental clip. Improvement in declaration could be achieved by cut downing the diffusion. An addition of the viscousness of the agarose gel reduces the diffusion, and declaration would therefore addition. The inauspicious consequence of this method is that it would decelerate down the experiment because the addition of viscousness of the agarose gel increases the clash of proteins. Increasing the experimental clip reduces the declaration and therefore is non ever a successful method to better declaration. This method would non be a good method for the proteins cytochrome C, myoglobin, haemoglobin, and serum albumen because Figures 2, 3, 4, and 5 show that haemoglobin and serum albumens are greater in size. That is, haemoglobin and serum albumens tend to be influenced by the clash. Another method to better the declaration is to increase the strength of the electric field. This method besides re duces the clip of the migration of the proteins. The lone 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 do the proteins to be denatured. Literature Cited Ravnskov, U. ( 1975, February ) . Low molecular weight albuminuria in association with paroxysmal myoglobinuria. [ Abstract ] Clin Nephrol 1975 Feb ; 3 ( 2 ) . 65-9. Retrieved January 31, 2001 from the WWW: hypertext transfer protocol: //www.ncbi.nlm.nih.gov/entrez/query.fcgi? cmd=Retrieve amp ; db=PubMed A ; list_uids=47277 A ; dopt=Abstract Caudie, C. , Allauzen, O. , Bancel, J. , and Later, R. ( 2000 March-April ) . Role of isoelectric focussing of cerebrospinal fluid Ig G in the early biological appraisal of multiple induration. [ Abstract ] Annales de Biologie Clinique. Vol. 58, Issue 2, 187-93. Retrieved February 2, 2001 from the WWW: hypertext transfer protocol: //www.ncbi.nlm.nih.gov/entrez/query.fcgi? cmd=Retrieve amp ; db=PubMed A ; list_uids=10760705 A ; dopt=Abstract Lunding, J. , Midgard, R. , and Vedeler, CA. ( 2000 Nov ) . Oligoclonal sets in cerebrospinal fluid: a comparative survey of isoelectric focussing, agarose gel cataphoresis and IgG index. [ Abstract ] Acta Neurol Scand 2000 Nov ; 102 ( 5 ) . 322-5. Retrieved February 2, 2001 from the WWW: hypertext transfer protocol: //www.ncbi.nlm.nih.gov/entrez/query.fcgi? cmd=Retrieve amp ; db=PubMed A ; list_uids=11083510 A ; dopt=Abstract Natural Toxins Research Center at Texas A A ; M University. Isoelectric concentrating. Retrieved January 29, 2001 from the WWW: hypertext transfer protocol: //ntri.tamuk.edu/if/if.html Bibliography Literature Cited Ravnskov, U. ( 1975, February ) . Low molecular weight albuminuria in association with paroxysmal myoglobinuria. [ Abstract ] Clin Nephrol 1975 Feb ; 3 ( 2 ) . 65-9. Retrieved January 31, 2001 from the WWW: hypertext transfer protocol: //www.ncbi.nlm.nih.gov/entrez/query.fcgi? cmd=Retrieve amp ; db=PubMed A ; list_uids=47277 A ; dopt=Abstract Caudie, C. , Allauzen, O. , Bancel, J. , and Later, R. ( 2000 March-April ) . Role of isoelectric focussing of cerebrospinal fluid Ig G in the early biological appraisal of multiple induration. [ Abstract ] Annales de Biologie Clinique. Vol. 58, Issue 2, 187-93. Retrieved February 2, 2001 from the WWW: hypertext transfer protocol: //www.ncbi.nlm.nih.gov/entrez/query.fcgi? cmd=Retrieve amp ; db=PubMed A ; list_uids=10760705 A ; dopt=Abstract Lunding, J. , Midgard, R. , and Vedeler, CA. ( 2000 Nov ) . Oligoclonal sets in cerebrospinal fluid: a comparative survey of isoelectric focussing, agarose gel cataphoresis and IgG index. [ Abstract ] Acta Neurol Scand 2000 Nov ; 102 ( 5 ) . 322-5. Retrieved February 2, 2001 from the WWW: hypertext transfer protocol: //www.ncbi.nlm.nih.gov/entrez/query.fcgi? cmd=Retrieve amp ; db=PubMed A ; list_uids=11083510 A ; dopt=Abstract Natural Toxins Research Center at Texas A A ; M University. Isoelectric concentrating. Retrieved January 29, 2001 from the WWW: hypertext transfer protocol: //ntri.tamuk.edu/if/if.html