2 replies to this topic

[NK22]

I am working on his tagged protein and using following buffers to protein elution through Ni-nta column
Binding buffer: 20mM Sodium Phosphate buffer , 500mM NaCl, 5mM Imidazole
Wash Buffer:20mM Sodium Phosphate buffer , 500mM NaCl, 30mM Imidazole
Elution Bufffer: 20mM Sodium Phosphate buffer , 500mM NaCl, 500mM Imidazole

the protein peak is not sharp during elution but very broad
Shall I increase the Imidazole Con centration in Elution Buffer?

[pesji]

NK22, on Oct 20 2009, 01:36 AM, said:

I am working on his tagged protein and using following buffers to protein elution through Ni-nta column
Binding buffer: 20mM Sodium Phosphate buffer , 500mM NaCl, 5mM Imidazole
Wash Buffer:20mM Sodium Phosphate buffer , 500mM NaCl, 30mM Imidazole
Elution Bufffer: 20mM Sodium Phosphate buffer , 500mM NaCl, 500mM Imidazole

the protein peak is not sharp during elution but very broad
Shall I increase the Imidazole Con centration in Elution Buffer?

No that won't help 500mM Imidazole is already more than enough You have to look on SDS PAGE where is really your protein don't forget that free Imidazole also absorb in the UV detector so the end of your pick might as well be free imidazole only !

[mdfenko]

you can stop the column flow for about an hour when the protein starts to elute to reduce the elution volume.

also, ensure that the previous solution has completely entered the gel before you start the elution buffer (to reduce initial dilution of the elution buffer).