In final steps of protein purification process , I fond out that after equilibration of q sepharsoe resin by this buffer, (20mM tris-HCl, 250 mM glycine, pH 7.4), final pH of resin after passing 10 CV of this buffer through the column wont change, and pH of out put of column will 8.5. PI of this protein is 5.6.
I want to applied a protein on to q sepharsoe column which is equilibrated by buffer (20mM tris-HCl, 250 mM glycine, pH 7.4,) and after capture of this protein by setting gradient 0-250mM NaCL, want to detach target protein from resin. protein was buffer exchanged in this buffer in advance.
For increasing the final specification of detach protein I want to apply this protein in a resin which is equilibrated by this buffer but in final pH 6.5, for avoiding Deamidation of protein.
In order to decreased final pH of column. could you please help me for designing a buffer for decrease final pH of resin after equilibration in order to reach pH 6.5?
Your question is rather confusing, but I think the simplest approach is this: Once you have eluted the protein from the column with NaCl in the original buffer, use dialysis to change the buffer to the final storage buffer. There are several choices of buffer to use at pH 6.5. Here is a web page from Sigma about buffers:
http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-reference-center.html
First, if you equilibrated your column with pH 7.4 buffer and you can not get the pH 7.4 after 10 CV washing the column with the equilibration buffer in the flow-through buffer pH, you can not load your protein, because the column is not equilibrated (the pH of the loading buffer should be exactly match the pH of the buffer after the column). If the column equilibration was OK, and this observation you described was only for the period of loading of the protein, you don't have to worry about that, that's normal ion-exchange processes in this type of chromatography. Wash the column as long as it takes to get the equal pHs to make sure that pH of of the buffer after the column is exactly matching the pH after the column.
Second, if you want to purify your protein more, I would suggest to use gel-filtration chromatography (which separates proteins based on their molecular weights) instead of other additional ion-exchange chromatography(s), and after that, separate (and probably purify) your protein with hydrophobic chromatography (which separates proteins based on their hydrophobic properties) on something like Phenyl-Sepharose or Octyl-Sepharose.
Third, if you want to decrease the pH of the final buffer to pH 6.5, you can do it any time with the buffer for gel-filtration or the buffer for your hydrophobic chromatography (but remember, that the closer your protein to it's isoelectric point 5.6, the higher it's tendency to aggregation and precipitation). Therefore, try to keep all your buffers for your protein purification at least 1 pH unit higher or lower than it's isoelectric point, where your protein starts to aggregate and precipitate.
Hope, it will be helpful.
@Viktor Y Butnev
thanks for your kind attention
For the first approach I should say that, I increased the volume of the buffer which is passed through the column to 20 CV but final pH of resin was not changed.
For your second approach I want to apply this process in industrial scale and as you know in this scale time of the process along with quality are most important factor for increasing benefit from a process, applying jel chromatography could increase the time of the process (I mean volume of loaded protein through the column, flow rate of this resin,...) as well as for getting best result for separation its more proper to use ion exchange insteasted of jel chromatography in order to increase quality of separation.
I want to apply this buffer (20 mM tris, 250mM Glycine, 50mM NaCL, pH 6) as a first buffer in order to over come the resistance of resin against pH then apply (20 mM tris, 250mM Glycine, pH 6.5) as equilibration buffer then applied protein on the column. the buffer of protein will exchanged in (20 mM tris, 250mM Glycine, pH 6.5) in advanced.
I know that this pH can increase the possibility of aggregation but I suppose that it is worth trying.
Hi Hadi,
The question seems rather distracting. If after CIP of Q-Sepharose by NaOH with a high pH or any other cases (pH=8.5) your equilibration buffer could not provide the desired pH (pH=7.4 or 6.5) for your processو simply you can apply a buffer with higher buffering capacity (e.g., 50mM Tris or higher) with the same components. Hope to be helpful.
@Hossein sedighikamal
thanks for your kind attention, after CIP process, I applied 20mM tris, 50mM NaCL in pH 6.5 then pass WFI through the column, Finally pH decreased to 6.5 then I applied eq buffer and elution buffer with the same composition as you know;) but in pH 6.5, by this approach fractionation was carried out in pH 6.5, then the stability of fractions increased to 3 month.
Hadi,
- I doubt whether pH=7.4 during the purification step (sample loading and elution) diamidates your protein of interest, because the temperature is not high enough, purification steps is not time consuming too much, and buffer components (glycine as a lyoprotectant and sink agent in the buffer).
- For anion exchange resins typically you need to work 1.0–1.5 pH units greater than PI of protein. This guarantees the optimum binding capacity.
- For your case a slightly acidic pH 6.3-6.5 is the best choice for a long-time storage of your elaute. pH adjusting of eluate (from 7.4 to 6.5) could be easily facilitate by addition of only few drops of an acidic agent with high buffering capacity (1000 mM Tris-HCl, pH~4) rather than changing the purification conditions.
Hope to be helpful. Best,
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