Sephadex - (Jul/10/2008 )

Our protein is 20 KDa protein and we use superdex 75 resin for gel filtration. but we are getting some high molecular weight contaminant bands too about 36-43 Kda. since the exclusion limit of superdex is more i was thinking of switching to sephadex G50 whose exclusion limit is perfect for my use.

but one problem is i cannot get research articles / application data sheet / paper where sephadex is used for gel filtration. eg packing sephadex, buffer to be used, running condition etc that will help me to plan my experiment.

i also visited amersham website but not much details on sephadex could be found.

if anyone has experience with sephadex handling kindly guide me.

thanks

How about something like a centricon with a molecular cutoff of 20 KD...

You put the sample in, spin it, keep what passes through... Molecules of a higher weight are trapped...

As for buffer, you can simply wet it with pbs, then put your normal sample in...

A COUPLE THINGS:

- Centricon is just one brand, there might be better ones out there now, it's been a while since I've used them.

- The molecular weight cutoff value is not absolutely.. it will let SOME molecules a little larger than 20 KD go through...

Is it possible that your protein is a dimer?

This is the manual I use (an oldie but goody)
http://www.amazon.com/Filtration-Theory-Pr...849&sr=11-1

Buffers should be same as you are using now. Might try to buy a prepoured column from GE life sciences or similar.
dan

Switch to low resolution resin G-50 is not a good idea. You can reduce that loading sample volume, elution flow rate and use less fraction volume to improve the separation.

Our protein is 20 KDa protein and we use superdex 75 resin for gel filtration. but we are getting some high molecular weight contaminant bands too about 36-43 Kda. since the exclusion limit of superdex is more i was thinking of switching to sephadex G50 whose exclusion limit is perfect for my use.

but one problem is i cannot get research articles / application data sheet / paper where sephadex is used for gel filtration. eg packing sephadex, buffer to be used, running condition etc that will help me to plan my experiment.

i also visited amersham website but not much details on sephadex could be found.

if anyone has experience with sephadex handling kindly guide me.

thanks