Verbatim_25

Dear all, First of all I have to say that I am not really experienced with cells and proteins isolation. My question is that I wanna isolated Histone H1 from a type of cells. There are a lot of reports, but all of them used acidic conditions (perchlorid acid, sulfuric acid, trichloroacetic acid...) and thats something that I would like to avoid. In some of the cases I understand that such conditions are used to precipitate the proteins and in others I guess that are to break/lyse the cell membrane nucleus. Are u aware of any method to isolate proteins from the nucleus avoiding acidic conditions? And second question, why such acids are being used to isolated such proteins (in case that my previous afirmations are wrong)? Thanks very much!

Hi People, Is it possible to label the N terminal of a peptide with Rhodamine B. As u know Rhodamine B has a carboxylic acid and under coupling conditions, that is HBTU/HOBt and DIPEA in DMF, could be easily incorporated. This is what I guess...but I wanted to ask whether u have any experience in the incorporation of this fluorophore to any peptide sequence. I didn't find any protocol in which is reported...and therefore I feel a little bit afraid about if it is possible to attach it... Thanks, Verbatim:25

Dialyse is one option, but it's time-consuming and I would prefer to use other faster techniques. MWCO filters is not really well-suited in my case since I am trying to purify one peptide that has 2000 Da as a molecular mass. It would also pass through the filter. I was thinking in Desalting Columns/buffer exchange which are faster than the previous ones.

Oh yeah. Tris when it's protonated in the amino group could compete for the anionic sites...I didn't get it before... By the way,my peptide is solved in Tris Buffer and I want to start the ion exchange separation with sodium phosphate buffer. The question is, how could I remove TRIS or how can I change the buffer? do u know any simple technique to do it? Thanks,

Hi People, I want to purify my peptide from other byproducts. I chose Ion exchange because my peptide (Fluorophore-KARK(modficiation)SAGA) its positively charged (Lysine and Arginine). I guess that the isoelectric point is around 11 and therefore cation exchanger is more suitable than anion exchanger. The thing is that my peptide is dissolved in Tris Buffer pH 8.2 and I would like to use it also as elution buffer (Tris buffer with NaCl). Nevertheless I read that for Cation exchanger is high recommended to use Phosphate buffer and Tris is more suitable for Anion exchange. My question is Why and if the use of Tris buffer represent any problem in the cation exchanger chromatography? Thanks,