If you want to purify a DNA-binding protein from a crude mixture of proteins at
ID: 147711 • Letter: I
Question
If you want to purify a DNA-binding protein from a crude mixture of proteins at pH 7, should you use a DEAE-celllose or a CM-cellulose column (see the figure below)? Explain your reasoning Weak anion exchanger (DEAE) Strong anion exchanger (Q) Weak cation exchanger (CM) Strong cation exchanger ('S") Match the items in the left column to the appropriate blanks in the sentences on the right. Reset Help DEAE At pH 7 a DNA-binding protein is expected to carry an excess ( lead to favorable binding to the highly charge that will charge on the DNA molecule (e.g., histones are charged).A -charged protein will bind to a column carrying an charge. Thus, at pH-7 celulose is expected to bind to a CM DNA-binding protein Keset heip DEAE At pH 7 a DNA-binding protein is expected to carry an excess ( charge that will lead to favorable binding to the ( charge on the DNA molecule (e.g., histones are charged). A charged protein will bind to a column carrying an )charge. Thus, at pH 7 cellulose is expected to bind to a CM DNA-binding protein. positivelyExplanation / Answer
1. Weak cation exchanger (CM)
Explanation: Proteins with positive charge can be separated using Carboxymethyl(CM) cellulose column with carboxylate groups because proteins with + ve charge at pH 7 binds with the carboxylate groups in weak cation exchanger of CM cellulose column. These can be then be obtained by using Sodium chloride in which sodium ions compete with proteins bound in the beads of column. Proteins with less density positive charge are eluted first which are followed by high density positive charge.
2. At pH= 7, A DNA binding protein is expected to carry an excess + (+ ve) charge that will lead to favorable binding to the - (negative) charge on the DNA molecule (eg histones are highly positively charged). A positively charged protein will bind to column carrying an excess (-)negative charge. Thus, at pH = 7, CM cellulose is expected to bind to a DNA binding protein.
Explanation: DNA is negatively charged due to phosphate groups. So, the DNA binding proteins should be positively charged. Inorder to bind positively charged protein column with negative charge is chosen. Carboxy methyl cellulose (CM) cellulose helps in separation of positively charged proteins while negatively charged proteins are separated using Diethyl Amino Ethyl Cellulose (DEAE) due to its positive charge.