Hi all. I try to purify a High molecular weight protein. I explain my case:
I did two purification with the same Culture and induction conditions. But when i purified the protein accidentally I change some buffer concentrations.
Buffer 1: Tris 50mM, NaCl 200mM, glicerol 10% and pH: 7,6
Buffer 2: Tris 20mM, NaCl 150mM, glicerol 10% and pH 7,6
Purification steps: HisTrap, MonoQ( the protein leaves in NaCl concentration 400mM) and Superose 6.
When I used the Buffer 1 I obtained a protein peak in 10,5mL of Elution volume but when i used the Buffer 2 I obtained two protein peaks in 9,8 and 11,3 ml Elution volume.
I change two concentration additives ( Tris and NaCl but I supposed that the NaCl concentration is the same in both cases, 400mM ( The NaCl concentration which the protein leaves in MonoQ).
Thank you.
From the context of your question, it is clear that you are referring to the Superose 6 chromatography. The only difference in conditions that you mention is the concentration of Tris (50 v 20 mM). This doesn't sound like a very big change, and I am inclined to think that the difference between the two experiments was due to some other variation between them.
However, one thing to remember about Tris buffer is that its pH changes a bit when it is diluted. So if you made a pH 7.6 buffer stock solution at 1M and then diluted it to 50 or 20 mM to prepare the elution buffers, the pH of the buffers may be a bit different. (Temperature also changes pH of Tris buffers). This difference will be slight, however.
Additionally, the more dilute buffer is more likely to be insufficient to prevent pH changes caused by the presence of other substances, such as concentrated protein. This could mean that the pH shifted significantly within the protein peak, and that could have an effect on protein-protein interactions.
Thanks for your answer Adam. Apart from Tris concentration The second buffer has 50mM less NaCl concentration than the first buffer.
The experiments are equal and I don't diluted the buffer.
Maybe Could it be the sample volume that I load in Superose6?
Hi David, are you using Tris base or Tris-HCL powder for the buffer?
The sample volume can have a large effect on the resolution of gel filtration columns. Larger sample volume reduces resolution. The two peaks you saw in one run could merge into one peak in the other run if the latter run had a substantially larger sample volume.
- Badges
- Science method