I want to purify protein and avoid RNase, so I try to use DEPC water to prepare a series of buffer, but the results show that the protein is not in a good state and tends to aggregate.
DEPC water needs to be incubated to allow reaction with proteins and autoclaved afterwards to remove DEPC. Only then should it be used. Otherwise the DEPC will react with your protein and destroy it.
Properly prepared DEPC water does not contain DEPC anymore and can be used to prepare buffers for protein isolation. If you are isolating the protein from a cellular source the use of DEPC water will however not prevent RNA degradation, because the main source of RNases would be the lysate.
DEPC (diethylpyrocarbonate) is a reagent commonly used to treat water and buffer solutions to remove RNases, which are enzymes that can degrade RNA. However, DEPC can also have an effect on proteins, and its use may cause protein aggregation.
DEPC can react with primary amines in proteins, such as lysine or arginine residues, forming a stable carbamate. This reaction may alter the conformation of the protein and make it more prone to aggregation. Additionally, DEPC may also cause oxidation of methionine residues and thus alter the protein's stability.
There are a few things you can try to minimize the impact of DEPC on your protein. One option is to use DEPC-treated water or buffer solutions that have been neutralized or de-activated. This can be done by adding a small amount of an amine, such as Tris or glycine, to the DEPC-treated water or buffer solution. The added amine will react with the DEPC, neutralizing it and minimizing its effect on the protein.
Another option is to use a different method for RNase removal. For example, you could use RNase inhibitors, such as RNasin, or RNase Away, which are enzymes that degrade RNase enzymes, and that is added directly to the buffer. These inhibitors can be added to the buffer before or during the protein purification step.
You also could try another method for purifying your protein, such as column-based purification like affinity chromatography, that can provide a high degree of specificity and purity, and avoid the use of DEPC-treated water.
It may be necessary to optimize the buffer composition and purification conditions to obtain the protein in a good state and avoid aggregation.
In summary, DEPC can cause protein aggregation by altering the conformation of the protein, it is important to neutralize DEPC-treated solutions or find an alternative method for RNase removal to avoid this problem and achieve a good protein purification.
DEPC water is used for nuclease-free reactions such as RNA DNA purification, while proteins used autoclaved afterward to remove DEPC because it degrades the protein.