Hello!
I am trying to do a ChIP experiment using the IgG sepharose beads from GE Healthcare. I work with a recombinant protein in Arabidopsis fused to TapTag (Calmodulin binding peptide, followed by tobacco etch virus proteasa cleavage site and Protein A, which binds to IgG).
I have some questions about the protocol (attached) provided with the beads:
* Why do I have to equilibrate the column (the beads in my case) with 0.5 M HAc pH 3.4? Does this step influence the bonding capacity of the beads? * Later, why do I have to wash the beads with 5mM NH4Ac pH 5.0? Why does the pH decrease in the wash?
Thank you very much,
Pedro de los Reyes.
Hi Pedro,
1- Why do I have to equilibrate the column (the beads in my case) with 0.5 M HAc pH 3.4? Does this step influence the bonding capacity of the beads?
--- Because you have to be sure, there is no other molecules which are linked to your beads (These beads contains IgG ligand which has affinity to your protein). with pH 3.4, you can get rid of all remaining proteins or other attached molecules in the column. This step exactly influence the bonding capacity.
2- * Later, why do I have to wash the beads with 5mM NH4Ac pH 5.0?
Ligand (IgG in your case) and your protein of interest interact each other with non-covalent bonding, so this binding occurs generally ionic binding. However, all proteins have some isoelectric points (pI) where the protein is neutr. So when you start your isolation process, pH is 7.6 and your protein and the other proteins all bind to ligand. When you decrease pH, you change the electrical charge of the all proteins. Your protein remains with ligand but the other just washed away. When you reach the no protein in your detector (I assume you use FPLC) in the washed tubes. You can elute or disconnect your protein of interest from the ligands.
Why does the pH decrease in the wash?
When you decrease the pH, you change your protein charge capacity and accordingly the interaction strength between the protein and ligand. like I said before all proteins have a certain isoelectric point (pI) at a certain pH (pI=pH) and they are neutr in this pH. Under or upper pH levels they become electrically charged. For example if pI of a protein is 7.6, when the pH 5, protein has 1+ charge, pH 3.4 this protein become 2+ positively charged.
pH 7 --- protein of interest (Poi) + other proteins are linked with ligand (IgG)
pH 5 ----- only poi remains in column -- other proteins (most of them) are washed away
pH 3.4 - Poi could be eluted from column
I hope this helps
Good luck
Cigdem
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