182, Gel Electrophoresis Experiment-Lab Manual Electric Force, Field and Potenti

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182, Gel Electrophoresis Experiment-Lab Manual Electric Force, Field and Potential in Protein Gel Electrophoresis Introduction Gel electrophoresi appropriate s is a well-established molecular biology technique that depends critically on application of physics principles. The goal of the approach is to separate components of ecular weight from a mixture of macromolecules in solution (c.g.DNA fragments or mixtures of proteins in a cell lysate) moving through a polymer gel under the influence of an external typical implementation, the separation of macromolecules by molecular weight is followed by an immunoblotting visualized via fluorescence or luminescence. At the end of this procedure, the intensity of that band procedure in which the protein bands are reacted with an antibody and relati ve to appropriate controls, can be interpreted as a reporter of the relative concentration of a particular macromolecule in the mixture that is being characterized (e.g. the level of expression of a certain protein relative to a loading control). Here, we focus on the first step of this procedure, the initial separation of macromolecules, which in our case will be a mixture of pre-stained proteins. In this procedure, called protein gel electrophoresis, a small amount of protein mixture is loaded into a polymer gel in a vertical gel tank filled with aqueous buffer solution. The buffer, which contains the detergent sodium dodecyl sulfate (SDS) and a reducing agent, denatures the proteins by breaking non-covalent bonds and causing proteins to unfold from their native three-dimensional conformations into linear chains of negatively-charged amino acids (Figure 1). In this important step, the SDS binds to the polypeptide core in a constant ratio of about one SDS molecule per every three peptide bonds. This imparts a constant (negative) charge per unit mass, such that the total charge, Q, of each protein is proportional to its molecular weight, M Treatment with SDS/ reducing agent Extended rod conformation coated with negatively charged SOS molecules Protein in native Figure 1:Schematic of the binding of SDS to a tightly folded globular protein (left) causing it to adopt an extended rod conformation with constant negative charge per unit mass (right) When a sufficiently a sufficiently large potential difference is established vertically across the gel, proteins migrate toward the anode at the bottom, slowly winding their way in a snake-like movement (called "reptation") through the microscopic network of pores in the polymer gel (Figure 2). cathode Direction of protein mgration gel anode Figure 2: A denatured protein migrating through the microscopic network of pores in a polyacrylamide gel (not drawn to scale).

Explanation / Answer

1. given
   potential differnece, V = 250 V
   gel length, l = 8 cm = 0.08 m
   so electric field inside the gel = E
   E = V/d = 250/0.08 = 3125 V/m

2. as force on the protiens with same charge is the same
   and for same force, acceleration is inversly proportional to the mass
   and distance travelled down the gel is proportional to acceleration
   hence distance travelled down the gel is inversly proportional to mass of protein
   so for three proteins, m1 , m2, m3
   distance travelled, d1, d2, d3
   d1 = k/m1, d2 = k/m2 , d3 = k/m3
   now, d2 = (d1 + d2)/2
   hence
   k/m2 = k(1/m1 + 1/m2)/2
   2/m2 = (m2 + m1)/m1m2
   m2 = 2*(m1*m2)/(m1 + m2)
   for m1 = 98, m3 = 62 we get
   m2 = 2*98*62/(98 + 62)
   m2 = 75.95
   hence mass of protein should be 75.95 kDa and not 80 Da

3. Vterminal for all the proteins is when drag force is equal to the electrostatic force
   this means v^2 would be proportional to electric field, and M is directly proportional to electric field ( higher mass particles experience more force)
   so square of vterminal and M would give us a linear graph

4. a. error due to scale measurement is instrument error
   b. this will effect velocity measurement as distance measured over period of time will have error due to instruemtn error of the scale
  

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