Re: [ccp4bb] CM Sephadex C50
Sounds odd as cross-linked resins should not exhibit such behaviour. I have seen this with e.g. phosphocellulose columns. The charged groups on the matrix repel each other at low ionic strength and expand hence the increased bed volume with water etc. At high ionic strength the charges are shielded and the bed contracts. This is not an issue with the most commonly used IEX beads these days as they are chemically cross-linked and resist the size changes. However, when I used to use Whatman P11 (phosphocellulose) even the changes in bed volume did not matter as it worked very well (in my case to bind phage T5 exonuclease). However, the bed volume changes do make the columns trickier to handle. Switch to a highly cross linked matrix like the rest of us! I now use GE Hitrap Heparin Sepharose as a good mechanically stable substitute. Cheers, Jon On 8 February 2017 at 06:47, syed ibrahim < 048c02cac012-dmarc-requ...@jiscmail.ac.uk> wrote: > Hello All > > I am using CM Sephadex C50 column for my protein. I equilibrated column > with MOPS buffer pH 6.0. But during elution I am using only NaCl2. This > looks like changing the bed height. Before starting elution the bed height > was around 60cm. After starting elution the bed height decreased and came > upto 12 cm only. After 1M NaCl elution I cleaned the column with ddH2O. The > column begin to enlarge to the height of more than 60cm. I dont understand > this behaviour. > > More over I could not find my protein as well. Similar situation arised > even if I change the buffer (Acetate buffer). > Any suggestions? > > Thank you > > Syed > -- Best wishes Prof. Jon R Sayers, FRSB Tel: +44 (0) 114 2159552 Email: j.r.say...@shef.ac.uk http://www.sheffield.ac.uk/iicd/profiles/sayers
Re: [ccp4bb] CM Sephadex C50
Yes, CM-Sephadex expands and contracts incredibly. For other than batch methods you will be much better using CM-Sepharose. I guess it is cross-linked sepharose, and doesn't swell up much even in distilled water. What is happening is the negative charges of the carboxy groups repel each other, causing the swelling. In high salt the charges are "screened" or neutralized by counterions (e.g. Na+). If you want to test your column, try with a colored protein. Cytochrome c sticks very tightly in 50 mM or less KPi 7.5, moves slower than the buffer between 50 and ~200 mM KPi, faster with increasing ionic strength. So you see a very tight band of bright red during loading, which diffuses and starts to move down during elution. On 02/08/2017 01:47 AM, syed ibrahim wrote: Hello All I am using CM Sephadex C50 column for my protein. I equilibrated column with MOPS buffer pH 6.0. But during elution I am using only NaCl2. This looks like changing the bed height. Before starting elution the bed height was around 60cm. After starting elution the bed height decreased and came upto 12 cm only. After 1M NaCl elution I cleaned the column with ddH2O. The column begin to enlarge to the height of more than 60cm. I dont understand this behaviour. More over I could not find my protein as well. Similar situation arised even if I change the buffer (Acetate buffer). Any suggestions? Thank you Syed
[ccp4bb] CM Sephadex C50
Hello All I am using CM Sephadex C50 column for my protein. I equilibrated column with MOPS buffer pH 6.0. But during elution I am using only NaCl2. This looks like changing the bed height. Before starting elution the bed height was around 60cm. After starting elution the bed height decreased and came upto 12 cm only. After 1M NaCl elution I cleaned the column with ddH2O. The column begin to enlarge to the height of more than 60cm. I dont understand this behaviour. More over I could not find my protein as well. Similar situation arised even if I change the buffer (Acetate buffer). Any suggestions? Thank you Syed
