Dear All, I am purifying my protein (theoretical PI = 8.9) in a buffer of pH =7.1. till Ni-elution I kept the DTT concentration (1mM). Usually, after elution people used to keep DTT concentration at 5mM. But in my until Ni-elution the protein look absolutely fine. But the moment I add DTT (5mM) thread like aggregate is formed. when I run those aggregate, it shows up as my protein. What could be the possibilities that DTT is making it precipitated. As I know DTT is used to solubilize protein as reducing disulfide bonds. I will appreciate your suggestions.
Thank you
With kind regards
Prem
Dear Prem
the effect of dtt and other reducing agent is not ever positive, it depends from the role of The cisteines, if the cisteines are involved in The formation of a structural s-s ( as for example happen in Cu,Zn sod1) the addiction of Dtt may induce protein unfolding and aggregation it precipitation. On the contrary if your protein contain just 1 cystene or the cisteines do not have any structural role but for examples are involved in The binding of a metal (as happen in Sco1) Then the addiction of Dtt can play a positive role.
you can find a more detailed explanation of it on my blog:ProteoCool (https://proteocool.blogspot.com/) in the presentation:
ProteoCool n°16
(3 Common mistakes in buffer preparation for protein ) at page 4
good luck
Manuele
Dear Prem
the effect of dtt and other reducing agent is not ever positive, it depends from the role of The cisteines, if the cisteines are involved in The formation of a structural s-s ( as for example happen in Cu,Zn sod1) the addiction of Dtt may induce protein unfolding and aggregation it precipitation. On the contrary if your protein contain just 1 cystene or the cisteines do not have any structural role but for examples are involved in The binding of a metal (as happen in Sco1) Then the addiction of Dtt can play a positive role.
you can find a more detailed explanation of it on my blog:ProteoCool (https://proteocool.blogspot.com/) in the presentation:
ProteoCool n°16
(3 Common mistakes in buffer preparation for protein ) at page 4
good luck
Manuele
DTT and Ni-metal affinity chromatography do not agree with each other.
For theory Manuele Martinelli's answer you can refer to. In practice I would first ask do you really need DTT? If your protein is already happy with buffer and salt you are providing I would recommend not to add protease inhibitors, BME, DTT. Keep the buffer minimal for better experiments.
To answer specific problem, check number of cysteine residues in your sequence and check how many of them are involved in disulphide bond formation. If there are no cysteine residues the reason could be something else. Check if there is any DNA binding site as last option if you rule out cysteine residues.
I personally would never add anything to buffer except salt unless specifically required. All the best
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