Based on th emolecular weight of the protein choose dialysis using dialysis bag or microsep centrifugation.

membranes for ultrafiltration and dialysis allowing the concentration and desalting of proteins with acceptable yields and little denaturing effect

Solid phase extraction cartidges work great and are much less time consuming than dialysis

Hi,

i agree with Diane Botelho; please use SPE kits.

http://www.merck-chemicals.com/life-science-research/proteoenrich-kits/c_5q6b.s1OqdEAAAEjqBp9.zLX

http://www.chem.agilent.com/en-US/Products/columns-supplies/samplepreparation/Pages/default.aspx

Regards

Selvan

Does salt required for stabilization of activity? or Did U precipitate with Ammonium sulfate or eluted with salt buffer? How much salt was there?

Dialysis a long process

Ultrafiltration takes less time but sample size in ml? Is it less than one ml with lowest concentration of 20Micrograms? Better use amicon filters. It takes just 10 min or less to get 20 microlits.

In my experience, the best and most convenient method is to desalt on small Sephadex 10 columns. For instance, the PD 10 columns from Pharmacia are most suitable. Usually, I desalt 1 ml protein solution (30 and more mg protein per ml). The column cannot become dry because of a porous plate on top of the column. After equilibration of the column with buffer, just add your 1 ml sample and let it penetrate the columns. When it has been completely soaked into the column, add buffer in 1 ml steps and collect each step in a tube. The desalted protein will appear after 3,5 ml. I usually take the 3,5, to 4,5 ml (or sometimes 5 ml) fraction. Salt comes at 5.5. to 7 ml. Pharmacia says in their instruction manual that you can desalt 3 ml and salt comes after 6 ml. This is risky. We have checked the desalting under various conditions and found that desalting 1 ml portions is optimal. The yield of the procedure described is ca. 70 %. However, it is fully active, because the metho is very gentle.

Column can take much time and produce more volume. Further it requires concentration

Dilution is about 1/3 . Which means that the concnetration instead of lets say 30 mg/ml is 20 mg/ml. I do not know our research but 20 mg per ml would suffice or most applications

A column can actually concentrate your sample too....I used to desalt urine on SPE columns...would start with 10 mL of urine and would elute in 0.5 mL so I actually concentrated my sample 20 fold.

Depends on what kind of column you are using. Cocentraion is only possible if you adsorb the protein to something like a matrix, in gel filtration it is scarcely possible.

Affinity column can concentrate protein. If salt requirement is not used for elution. Then affinity is best.

Hi Avani. You can use ZipTip from Millipore http://www.millipore.com/catalogue/module/c5737

ZipTip is useful if you're dealing with very low volumes of protein. It desalts and concentrates your sample. It is very useful if you want to do some mass spec analysis and need to get rid of any salt before doing any sensitive downstream experiments. Hope this helps. Good luck! :)

1. zip-tip is the ideal method...

2. we follow this protocol, the best I have ever come across: http://www.sciencedirect.com/science/article/pii/0003269784907826

3. try TCA/Acetone or just acetone precipitation. protocols are available even on google.

here salts are used. detergent is another problem. Trailing can be observed on PAGE in salts and detergents.

http://antibody-antibodies.com/search_full.php?search=geba flex

with gebaflex columns by electoelution or reverse osmosis. How large is your protein?

We use two methods... One is the FASP method from the Mann group, which utilizes molecular weight cut-off filters, and we also use a Reverse Phase column like a Zip-Tip. I think that generally the Zip-Tip is the easier option.