How can we make a crystallizing buffer at basic pH while the acidic PEG solution decreased the final pH of the solution? We optimized a crystallizing solution from 0.1M SPG buffer+25%PEG1500 in PACT-Qiagen screen kit. We made 1M SPG buffer stock pH 9 then combined with PEG1500 in order to vary percent PEG in grid screen but the pH of the final solution is much lower than what we would like it to be. Could anyone suggest how to tackle this issue please? We need the final pH to be close to 9 as observed from the PACT-Qiagen screen kit.
Have a look at Arnold et al, JMB 2007 (PubMed ID 17217961). There, we used a mixed-bed ion exchange resin to pre-incubate PEG stock solutions - BEFORE adding them to buffers. Its in the Methods section, under "Electrophysiology".
PEG, through oxidation in contact with air/oxygen, can form organic acids that are absorbed by the resin. Make sure that no resin is added to your buffers when adding PEG from stocks to your buffers. It also helps to use high-quality PEG, and to make PEG solutions fresh rather than keeping them on the bench for ages.
Could you please tell me what is the volume of the drop? As I know I should not be change because the job of your buffer is to keep the pH.
As you can see in the PACT suite in pH. 9 it does not need to add salt, then if you mix 0.1 M SPG buffer pH. 9 with 25% PEG 1500 in each drop it should be fine. The final pH. Will be 9.
P.S. don’t mix the 1 M stock solution with PEG 1500 at first.
could you please tell me what is the valume of the drop? as I know i should not be chang becuse the job of your buffer is to keep the pH.
There is a more subtle way of changing the buffer while keeping the chemicals the same without using alot of NH3 and NH4CL because the composition is :
SPG buffer is produced by mixing succinic acid, sodium dihydrogen phosphate,
and glycine in the molar ratios 2:7:7 – succinic acid:sodium dihydrogen phosphate:glycine
You can reduce dihydrogen phosphate and replace it with monohydrogen phosphate which has a pKa above 12. Say something like 5 part dihydrogen and 2 parts monohydrogen this will shift the pH to basic without changing phosphate concentration.
Mixing these two phosphates to make a phosphate buffer is an old trick which allow a wide range of pHs without needing to titrate with NaOH. There are tables which give the ratio of the two which produce the desired pH. The benefit of this is that you will have a buffer as close to SOG as possible but slightly more alkaline. I think that the problem with SPG in PACT is that only the Glycine in it is good at pH9 as it has a pKa of 9.6 so the only other solution is to add more Glycine but that will change the molecular ratio composition of the buffer which you may not want..
Thanks a lot for all useful suggestions here.
@Dirk: Is DOWEX the choice for an ion exchange resin in PEG solution? Please suggest. (I am sorry to ask since at the moment I do not access to the paper in JMC2007 you suggested.)
you need to use a mixed-bed anion/cation exchanger. We used Amberlite IRN-150 mixed-bed ion-exchange resin at the time. Its convenient because the beads are big and easy to filter, but I'm sure other resins will do as well
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Materials Chemistry
- Polymer Chemistry