Elution of His-tag Protein under denaturing condition - (Aug/21/2007 )
Hi, i`m new in this forum and want to ask you about the purification using the Ni-NTA beads from Qiagen. In my project i want to overexpress a 17kDa Protein(incl. His tag) using e .coli. And i know that my protein will come as Inclusion body, because of the high expression of the protein production, that`s why i use the purification under the denaturing condition.
But after binding with Ni-NTA beads i have a problem to elute all of my protein from the beads, since there are still a lot of proteins that stuck on the beads after i run the SDS -Page gel. To solubilize my protein i use the 8M Urea, Tris, and NaH2PO4 with pH 8.0 and to wash i use the same Buffer but with pH 6.3 and to elute my protein i use the same Buffer with pH 4.5, and i have tried before to use a different Ph Concentration like 4.0;4.2;4.5;4.6; and 4.8 but it doesn`t work, using all of this pH value can elute only some of the protein but not all of them from the beads. I have also tried to reduce the volume of the beads but it doesn`t work too.
I hope somebody can help me to solve this problem. Thank you
But after binding with Ni-NTA beads i have a problem to elute all of my protein from the beads, since there are still a lot of proteins that stuck on the beads after i run the SDS -Page gel. To solubilize my protein i use the 8M Urea, Tris, and NaH2PO4 with pH 8.0 and to wash i use the same Buffer but with pH 6.3 and to elute my protein i use the same Buffer with pH 4.5, and i have tried before to use a different Ph Concentration like 4.0;4.2;4.5;4.6; and 4.8 but it doesn`t work, using all of this pH value can elute only some of the protein but not all of them from the beads. I have also tried to reduce the volume of the beads but it doesn`t work too.
I hope somebody can help me to solve this problem. Thank you
Hi,
better you try to elute your protien with 300mM imidazole in elution buffer. you can add 10mm and 20 mm imidazole in your lysis and wash buffer respectively to prevent nonspecific binding.
good luck
But after binding with Ni-NTA beads i have a problem to elute all of my protein from the beads, since there are still a lot of proteins that stuck on the beads after i run the SDS -Page gel. To solubilize my protein i use the 8M Urea, Tris, and NaH2PO4 with pH 8.0 and to wash i use the same Buffer but with pH 6.3 and to elute my protein i use the same Buffer with pH 4.5, and i have tried before to use a different Ph Concentration like 4.0;4.2;4.5;4.6; and 4.8 but it doesn`t work, using all of this pH value can elute only some of the protein but not all of them from the beads. I have also tried to reduce the volume of the beads but it doesn`t work too.
I hope somebody can help me to solve this problem. Thank you
Hi,
better you try to elute your protien with 300mM imidazole in elution buffer. you can add 10mm and 20 mm imidazole in your lysis and wash buffer respectively to prevent nonspecific binding.
good luck
Thank you for your reply, i`ll try it. I appreciate your help