Hi,

It is my understanding that you want to purify your protein from a number of proteins of similar sizes.  I would not recommend using a sephadex resin for this.  Sephadex resins are typically used for desalting and group separations, where you are separating molecules that vary greatly in size: it will not have the resolution you need to separate proteins that are running closely together on a gel.  In order do that, you will need to select a size exclusion chromatography resin that is meant for high-resolution separations.  I recommend Superdex 200, Sephacryl S100 or Sephacryl S200 resins for this specific purification, as these resins are designed for effective resolution of molecules near your protein of interest’s molecular weight.  If you are new to size exclusion chromatography, I suggest using a long column with a narrow diameter to optimize resolution, and keep the sample loading volume between 1-3% of the column volume.  I would also advise you to use a calibration kit before loading your sample, so you can familiarize yourself with your column’s performance, and experiment with loading volumes and flow rates to get the best separation for your needs.

Good Luck!

I will suggest you to use sephadex g100 as its Fractionation Range is 4-150 KDa. Sephadex g50 has range from 1.5- 30 KDa where your protein will be in void volume, on the other hand g75 has range from 3-80 KDa which is very close to your protein's molecular weight.

I will also suggest you to use SF grade for best resolution.

Please see the attached paper where the authors have used sephadex g100 to separate 60KDa protein.

Article Spermidine-Binding Proteins. Purification and Expression Ana...

Dera Himanshu:

I suggest you to use G75. The longer the column is, the better resolution you will get (but in this case, your protein sample should be high because the process will dilute protein concentration, and consequently, you will have problems to detect it after all).

Sephacryl resins are also appropriate.

Good luck,

Rosa María

Dear Sharma

your best choice is sephadex G100 this will return your protein and other smaller proteins in your protein mixture try to use longer column to allow better separation.you also can try sephadex G50 in this case your protein will hopefully will be eluted in the void volume while other proteins smaller than your protein will be stick in the column by this trick you might have your protein pure in the void volume here you can concentrate it. Good luck

http://labs.mcb.harvard.edu/Gaudet/Resources_Files/GEHealthcare_chromatography/Don't%20move/18102218.pdf

Please find the Gel Filtration manual for free and look for your best choice. It is easy, you can find a table with the separation data.

Best regards,

Ana Maria

Hi,

It is my understanding that you want to purify your protein from a number of proteins of similar sizes.  I would not recommend using a sephadex resin for this.  Sephadex resins are typically used for desalting and group separations, where you are separating molecules that vary greatly in size: it will not have the resolution you need to separate proteins that are running closely together on a gel.  In order do that, you will need to select a size exclusion chromatography resin that is meant for high-resolution separations.  I recommend Superdex 200, Sephacryl S100 or Sephacryl S200 resins for this specific purification, as these resins are designed for effective resolution of molecules near your protein of interest’s molecular weight.  If you are new to size exclusion chromatography, I suggest using a long column with a narrow diameter to optimize resolution, and keep the sample loading volume between 1-3% of the column volume.  I would also advise you to use a calibration kit before loading your sample, so you can familiarize yourself with your column’s performance, and experiment with loading volumes and flow rates to get the best separation for your needs.

Good Luck!

I  suggest you to use sephadex G 75. It will help to you for separation of your protein. Then you will collect elution and performed SDS PAGE trhough silver nitrate procedure. This method is easyly detected your protein on the gel. 

thank  you all for the suggestions.............................