Please help me understand the information provided . How can I interpet the data
ID: 132650 • Letter: P
Question
Please help me understand the information provided.
How can I interpet the data in the three graphs shown? How do I determine which is the contaminent and which is my protein of interest?
Your protein sample has two proteins in it (Protein gel, "Pre" sample). One is a contaminant; the other is your protein of interest (~50 kDa protein) You take your sample and put half over a sizing column, and half over an anion Sizing column exchange column SDS-PAGE : Sizing : Anion Da L Pre: A B A B 75 50 25 Anion exchange column (Elution by salt gradient) Ecation of Ekution Sample 025 I Protein A 0.00 925 A 0.00Explanation / Answer
From the data given, the protein in column A is protein of interest, and the protein in column B is contaminant
When we consider sizing column, the largest proteins will first elute and smallest last . And SDS PAGE shows protein of interest is ~50 kDa protein
When we consider, anion exchange then salt gradient is used to elute separated proteins. When low salt concentrations, then proteins having few charged groups are eluted and when higher salt concentrations, proteins with more charged groups are eluted.
Unwanted contaminants minimize elution so takes more regeneration time as in column B