isoclectric focusing. In a second step, a strip of this gel is turned 90 degrees
ID: 206774 • Letter: I
Question
isoclectric focusing. In a second step, a strip of this gel is turned 90 degrees, placed on another gel containing SDS, and electric current is again applied. In this second step: A) proteins with similar isoelectric points become further separated according to their in two-dimensional gel electrophoresis generates a series of protein bands first step molecular weights. B) the individual bands become stained so that the isoelectric focus pattern can be visualized. C) the individual bands become visualized by interacting with protein-specific antibodies in the second gel. D) the individual bands undergo a second, more intense isoelectric focusing E the proteins in the bands separate more completely because the second electric current is in the opposite polarity to the first current. 7. For each of these methods of separating proteins, describe in detail the principle of the methods, the physical and experimental parameters influencing the strength of interactions between the protein and the columns and the elution of the proteins. (20 points) (a) ion-exchange chromatography (b) size-exclusion (gel filtration) chromatography (c) affinity chromatography 8. A biochemist is attempting to separate a DNA-binding protein (protein X) from other proteins in a solution. Only three other proteins (A, B, and C) are present. The proteins have the following properties: (10 points) (isoelectric Size Bind to DNA? protein A protein B protein C protein X 82,000 21,500 23,000 22,000 7.9 What type of protein separation techniques might she use to separate: (a) protein X from protein A? (b) protein X from protein B? (c) protein X from protein C? 9. As a protein is purified, both the amount of total protein and the activity of the purified protein decrease. Why, then, does the specific activity of the purified protein increase?Explanation / Answer
6. A- Proteins with similar isoelectric points( pI) become further separated according to their molecular weights.
2-DE ( two-dimensional gel electrophoresis is a tool to compare the proteomics. 2-DE migration of proteins are separated by charge -isoelectric point in the 1st dimension and further separated by mass in the 2nd dimension on 2-D gels.
7. a. Ion- exchange chromatography is for separtion of proteins. It provides high resolution separation of proteins with same sign but various total net charge.
Most proteins bear nonzero net electrostatic charges at all pHs except at pH-pI( isoelectric point)
Ion -exchange chromatography is due to electrostatic attraction between buffer- dissolved charged proteins and oppositely charged binding sites on a solid ion-exchange adsorbent.
The ion-exchange adsorbent is placed into a column, protein mixture is applied onto the column. Proteins are charged oppositely to ion-exchange media are temporarily retained in the column nd all other proteins pass through the column are collected.
The retained proteins are eluted from the column by applying a modified buffer by increasing the ionic strength of the buffer.
b. Size exclusion chromatography also known as gel flitration chromatography is for separation of protiens on the basis of their size. It does not depend on the chemical interaction with the proteins. It is purely based on a physical property of the protein- the size.
separation is achieved by the differential exclusion or inclusion of solutes as they pass through the stationary phase consisting of pores of different sizes cross linked polymeric gels an beads.
large proteins cannot enter these pores pass on the outside of the beads. So the volume of the column appears smaller to the large molecules.
Smaller proteins can enter the pores of the beads. Both smaller and larger proteins experience the same flow rate.
The big proteins elute forst and the small proteins elute last.
c. Affinity chromatography: It makes use of specific affinity between a substance to be isolated and a molecule that it can specifically bind.
It has a matrix and a spacer arm.
i. The matrix should be physically and chemically inert .
ii. The spacer arm is used to improve binding between ligand and target molecule. The ligand is the molecule that binds reversibly to a specific target molecule and enables the purification by affinity chromatography and to perform this it shpould have chemically modifiable groups that allow it to be attached to the matrix without destroying the binding activity.
8. Separation of protein x from protein A can be done based on the size-exclusion chromatography as both proteins have similae pH( 7.4- 7.8) and both have an affinity to DNA (both are bound to DNA). But their size differs. (The protein A 82.000 and protein x has 22,000)
Separation of protein X from protein B can be done by iso-electric focus on ion-exchange chromatography because protein B has pI of 7.8 and protein X has pI of 3.8 and both have an affinity for DNA and both have similar size.
Separation of protein X from protein c can be done by affinity chromatography as both the proteins have similar size and pI( iso electric point) but protein X is bound to DNA where as protein C is not bound to DNA.
9. The total activity of the protein and the amount of protein during purification decreases because during purification process, the protein is lost at each step and also its activity decreases because it gets denatured during manipulation.
Specific activity is the ratio of activity units to amount of protein (U/mg) which should increase during purificTION. During purification process, lots of undesired proteins aare purified away, but the desired protein giving the activity remains thereby becoming enriched at each step. So the rati of total activity to total an=mount of protein increses.
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