My protein got precipitated during overnight dialysis. Is 150mM NaCl too less for Dialysis buffer of 500ml? And i used 20mM Tris and 10% glycerol.
If your protein precipitated then your answer is yes. Every protein is different and may have highly varying stability. If you need it to be in lower salt, then try some other stability buffers (different ph, more glycerol, add sugars//trehalose/sucrose, change buffering system). Avoid cold as that will increase the likelihood of precipitate. If this answer doesn't help, let me know what your goals are and I can give more specific advice.
If you were dialyzing protein out of a denaturing buffer the protein will become insoluble and fall out of solution. If this is the case, you may need to dialyze into lower concentrations of the denaturant, or you may need to try a refolding kit.
Hi there,
Is NaCl the only varying ingredient between initial and final buffers?
I am thinking to try with Phosphate buffer system (initially I tried with Tris), and also to increase NaCl concentration to 500mM to trouble shoot.
my protein also precipitated by dialysis. But funny is it can survive PD10 desalt column. maybe it works by you too then it can really save time and materials.
Increase to 50mM the buffer concentration, ph 7 or 7.5 (the pH should be dependent of the kind of protein), the NaCl conc. (150mM) is close to that of the blood serum, why?
Before you do anything dramatic, what is your goal in attempting to lower the salt? Do you need a lower NaCl concentration and why? Dr. Liger and I are asking the formulation questions to better gauge what you should try first.
From what I know NaCl helps to prevent any aggregation of the protein, thus, I considered to increase the NaCl concentration to 500mM in Tris buffer, so as to prevent any precipitation. Initially, I used 150mM NaCl with 10% glycerol and Tris buffer.
That is correct that NaCl may help prevent aggregation. But why do you need it more diluted? What is your end use for this protein?
I don't understand your question. Your protein is in 500mM NaCl and Tris, and if you dilute your sample then you will dilute your protein as well. The glycerol is just as likely to alter your enzymatic assay as the 500mM NaCl. Keep in mind that PBS, and "normal" biological activity, is equal to 144mM salt. I cannot tell you whether your enzymatic assay will be affected by 500mM, but usually no. Also, I am slightly concerned about your statement. If you have measureable protein, then you have a high protein concentration that will likely be diluted when you run your bioassay. So before you start running all of these extra experiments you should probably test what you have. You should also read the protocol for your enzymatic assay as it will tell you if glycerol or salt is inhibitory.
Also, keep in mind even if the salt is inhibitory, you can put your standards in the same salt/glycerol concentration to account for the variation.
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