My protein is 28kda and is some what hydrophobic with a pI of 6 and has a his-tag. We typically purify in 1x TBS with low salt at pH 7.5. During dialysis and or concentration the protein precipitate and majority is lost? One of my questions is can I purify in no salt buffer? What else can I do to improve my yield and avoid precipitation?
Hi Ranjuna, we used to have a similar problem with our protein. Have you added RNAse at any step? This may prevent RNA binding to your protein, causing it to conglomerate into rna-protein complexes and crashing out of solution.
Hi Ranjuna,
Read this article, they discussed very well about how to overcome the protein precipitation.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5368116/
Can you please mention the salt concentration you are using for protein purification? I would suggest using 150-300mM NaCl, also 5-10% glycerol in the buffer might help.
If nothing works, you may have to use 8M Urea for protein purification
To answer your question, you should not purify protein in no salt buffer. The solubility and stability of proteins usually increases slightly in the presence of salt. I would recommend you to use around 200-250mM NaCl in your dialysis buffer as mentioned by Dr. Papri Basak. Increasing your salt concentration might solve your problem. Is your protein a membrane one? Membrane proteins tend to form aggregates.
Thank you everyone for your input. The current salt concentration I'm using is 75mM, we have noticed that higher salt concentrations tend to crash the protein as well.
Yes, this is a membrane protein.
We have tried SEC on the protein as well, the result was the loss of protein. I should also mention that my protein has a tendency to bind agarose columns.
I'm also considering using a desalting column at this point.
Papri Basak thank you for your suggestion. I'm not familiar with the use of glycerol. Generally speaking, what is the effect is on proteins. How can this help with purification and reduce precipitation?
Glycerol increases the stability of proteins. You can use about 5% glycerol in your dialysis buffer.
One reason that your protein is getting pelleted/precipitated is that your protein has not folded properly when overexpressed and is not soluble enough leading to aggregation (as it's a membrane one). If your problem persists you can try & purify the protein under denaturing conditions & then refold it.
Hi Ranjuna,
I agree with Tushar Chakraborty. According to my previous experience, glycerol can indeed effectively stabilize the properties of a protein. By the way, is your protein truncation? If the truncated region is not selected properly, it is common that the expressed protein cannot be folded correctly.
Good luck!
@Ranjuna Weerasekera 5% glycerol helps in stabilization of protein. They don't affect activity of the protein isolated, instead degradation is reduced after freeze thaw.
For isolation of membrane proteins, as earlier, I would suggest using urea (denaturing condition). Don't worry about the activity, works fine wih enzymes as well. Try it. Best of luck!
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