During the final stages of the process: when the peptides are being desalted and concentrated with C18 zip tips (Thermofisher C18 Tips, 10 µl), followed by vacuum evaporator (speed vac), we get the picture of the white spots on the bottom of the test tube. It is implied that the white spots/areas are the Urea which wasn’t removed during the previous steps. To verify this, we conducted a mock experiment using only Urea dissolved in 0,1M Tris-HCl and repeated the steps pretending as if it is a sample with peptides. It resulted in the same output = white spots.
After that we tried to remove the Urea by adding Ice-cold Acetone to pellet the Urea and get rid of it by washing it with acetone again, however, in the end, we still got the impure peptides and in small quantities.
Can somebody suggest a solution?
To remove urea and sarkosyl during sample preparation (in-solution), one approach is to use gel filtration. The buffer volume used for extraction should be 0.5 to 1.0 ml per gram wet weight of original cells if the extract will be subjected to gel filtration
Based on the information you have provided, it seems that the white spots/areas you are observing are indeed due to residual urea that was not removed during the previous steps. Urea is a highly soluble and polar compound, and it can be difficult to completely remove from peptide samples. The addition of acetone can help to precipitate urea out of the solution, but it may not be sufficient to completely remove all of it.
One possible solution to this problem is to use a different desalting and concentration method that is more effective at removing urea. For example, some researchers use solid-phase extraction (SPE) cartridges with specific sorbent materials to remove contaminants like urea from peptide samples. Alternatively, you could try using a different type of C18 tip or a different brand of tip that may have better urea removal capabilities.
Another option is to consider changing the sample preparation protocol to remove urea earlier in the process. For example, some researchers use urea-free sample preparation methods, such as filter-aided sample preparation (FASP), which can reduce or eliminate the presence of urea in the final sample.
As you know Urea and Sarkosyl are two common contaminants that can be difficult to remove during sample preparation. Here are some methods that can be used to remove these contaminants from protein samples in solution:
Dialysis: Dialysis is a widely used method for removing small molecules like urea from protein samples. It involves placing the sample in a dialysis membrane and allowing the small molecules to diffuse out of the membrane into a buffer solution. Dialysis can be time-consuming, but it is effective for removing urea and other contaminants.
A. Gel filtration chromatography: Gel filtration chromatography (also known as size exclusion chromatography) separates proteins based on their size. This technique can be used to remove small molecules like urea from protein samples. The sample is loaded onto a column containing a gel filtration resin, and the proteins are separated based on their size. The small molecules are excluded from the resin and elute in the void volume, while the larger proteins are retained and elute later.
B. Precipitation: Urea and Sarkosyl can be removed by precipitation using organic solvents such as acetone or ethanol. The protein is precipitated by adding the organic solvent to the sample, and the precipitate is then collected by centrifugation or filtration. This method is simple and fast, but it may not be suitable for all proteins.
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C. Filtration: Urea and Sarkosyl can be removed by filtration using a filter with a molecular weight cutoff that is smaller than the protein of interest. The small molecules pass through the filter, while the larger proteins are retained.
It is important to note that the method used to remove urea and Sarkosyl will depend on the specific protein and the downstream application. It is always advisable to optimize the sample preparation method to ensure that the protein is free from contaminants and suitable for the intended application.
hope my answer helps us. good luck
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