Why exactly do you need to remove the Coomassie blue? What would residual Coomassie blue do to your immunization?

A wash with ammonium bicarbonate will be useful. The removal of CBB van be achieved by the buffer salt ammonium bicarbonate (NH4HCO3) and a fraction of 30%-50% organic solvent acetonitrile. The hydrophobic interactions between protein and CBB are reduced by the acetonitrile fraction. At the same time diminishes the ionic part of the solution the electrostatic bonds between the dye and the positively charged amino acids of the protein. In contrast to a mixture of water with organic solvent the effectivity of destaining is increased. To a certain degree (< 10%) the destaining procedure is accompanied with a loss of protein.

I just assumed that the CBB would be undesired in the immunization. If it is not a problem then that is not hte main problem. I do not want to purify the protein with precipitation because of the risk of loosing much sample.

the two ways you suggested are the best options with fewer loss of sample, consider the cut-off on the amicon filters, as you are going to lose everything below that, probably the best option would be a desalting column. I assume the coomassie blue is both in solution and attached to the protein though... You're going to get rid of the colorant in solution, but the part attached to the protein will remain... An option might be, if the desalting column or the amicon filters are compatible with solvents, to resuspend your protein extract in destaining solution (Methanol or Ethanol, acetic acid and water sorry i don't remember the proportions now), and filter it through, this should work better with the amicon filters i guess... Let me know how you get through :)

Why removing it since it may not have any impact positively or negatively on the immunization process. However, using NH4HCO3 and a fraction of 30%-50% organic solvent acetonitrile will be suitable for the removal.

Prafull's, Massimiliano and Henry's answers are insightful. Cannot add anything better !! :-)

just remembered something, i assume that since you are using this protein for immunization you have a high concentration of it. Instead of going crazy for the destaining that however results in a small sample loss, from next time you can use a colloidal coomassie staining that is way easier to remove as it doesn't penetrate in the gel, even if it's less sensitive though, now I don't have it on my fingertips, but if you think it can be useful let me know and I can find it for you.

Cheers,

max

Thanks Prem. Good luck Anette in your work.

Thank you all for the advice. I decided to go with Massimilianos advice to dilute the sample in destaining buffer and change buffer with the Amicon ultra device. Most of the stain was adsorbed to the filter, but so did much of the protein as well. The sample loss was large, but I obtained sufficiently for the immunization. Next time I will probably use some other staining so thank you for telling me about those.