11. Mixture of 4 proteins is subjected to anion-exchange chromatography (AIEX) a
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Question
11. Mixture of 4 proteins is subjected to anion-exchange chromatography (AIEX) at pH 7.0 Information about proteins are given in the following table: Ri i | Net charge at pH 7.01 (-+, neutral) Proteins bind to AIEX (YES or NO) Protein pl Mm (kDa) from AIEX A 9.8 B 5.6 C 2.2 25 80 50 15 (a) Determine the net charge for each protein at pH 7.0? Which proteins will bind to AIEX matrix? (Fill the table) (b) What is the order of elution from AIEX? (Fill the table). (e) Is it possible to separate prot ins Cand D using AIEX chromatography? Explain your answer. (d) When proteins C and D are subjected to gel filtration (GF) chromatography, which protein will be eluted first? (e) Mixture of proteins A,B,C and D was analyzed by 2D electrophoresis (proteins are first separated by isoelectrofocusing based on the pl, followed by SDS PAGE) and the following gel was obtained after staining:Explanation / Answer
Answer:
AIEX (Anion Exchange column)
Anion exchange chromatography uses resin that contains positively charged groups such as diethyl-aminoethyl groups (DEAE).When a mixture of protein sample is used along with anion exchange resin, then the most negatively-charged protein will be most attracted to the stationary phase. As a result, when the anion exchange chromatography is performed, the protein that carries the most negative charge will elute last and the protein that carries the highest positive charge will elute first.
Proteins C and D have a very narrow difference in pI, therefore they will not resolve or separate very well in a anion exchange chromatography.
When proteins C and D are subjected to gel filtration chromatography then protein C will be eluted first. This is because the MW of protein C (50 kDa) is more than the MW of protein D(15 kDa). In size exclusion or gel filtration chromatography, the higher size or molecular weight molecules are eluted first.
Protein pI Mm (kDa) Net charge at pH 7.0 (-,+, neutral) Protein bind to AIEX (YES or NO) Order of elution from AIEX A 9.8 25 +ve No Will not bind, and elute in void B 5.6 80 -ve Yes 1st C 2.2 50 -ve Yes 2nd D 2.0 15 -ve Yes 3rdRelated Questions
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