Examples of elution profiles are represented bellow. The red line in A correspon
ID: 143104 • Letter: E
Question
Examples of elution profiles are represented bellow. The red line in A corresponds to the conductivity signal (related to ionic strength of buffer). Answer the following questions about these purification assays.
1)Which protein has bigger pl ?Explain
2)Which peak (IgG purified) would you keep in assay B?
Column Protein A Sepharose Fast Flow XK 16/20, bed height 4.8 cm (9.6 mL) clarified cell culiture containing lG2 Sample: 500 ul of T. reesei crude cellulases Column Mono QT HR 5/S Buffer A 20 mM Tris-HCI, pH 76 in buffer A, 2.5 mg Sample volume: 600 mL containing 87.6 mg Starting buffer 20 mM sodium phosphate, pH Elution buffer 20 mM sodium citrate, pH 40 Flow Flow1.0 ml/min Buffer B A+0.5 M NaCI 5 mU/min (150 cm/h) Gradient 0% B for 4 min, 0-40% in 21 min. 40-100% B in 15rmin 0.5 10 0 10 20 30 Time (min) 200 Fig 1. Examples of Elution profile of purification processes taken from Protein Purification Handbook (Amersham Pharmacia Biotech)Explanation / Answer
An elution buffer plays an essential role in every immunoprecipitation protocol or assay that requires the release of a target antigen from a capture antibody. Elution buffers are necessary in protocols utilizing a stationary affinity column, and are also required in protocols using mobile solid supports in solution.
The elution buffer interferes with and disrupts antibody-antigen interactions. The premise of an immunoprecipitation assay is that capture antibodies are immobilized on a support platform and specifically bind target antigens. These antibody-antigen complexes are then recovered either by centrifugation or magnetic separation, but the antigens need to be separated from the antibodies for examination by SDS-page. If the antibodies remain bound to the antigens then they will contaminate the SDS-page and complicate analysis by Western blot.
Elution buffers are chosen for their ability to interfere with ionic, hydrophobic, or hydrogen bonding.The most common method of interfering with ionic interactions is to use an ionic elution buffer with a high salt content. NaCl solutions are often used. The pH of the buffer can also play a role as protein interactions are most efficient at physiological pH. Raising or lower the pH can help disrupt those interactions. One of the gentlest and most commonly used components in non-denaturing elution buffers is glycine-HCL with a very acidic pH of 2.5-3.0. This buffer disrupts the antibody-antigen interactions without denaturing the proteins. After the antigens are collected the pH is neutralized to prevent long-term damage to the proteins.
Solution:Crude cellulase will have a more pI as the buffer used to elute the same is of basic nature(elution buffer should have a pH which is greater than the pI of the protein and pH of the starting buffer.
peak (IgG purified) : volume at 600 units
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