Hello, I have been using cation exchange (HiTrap SP) for my purifications on ATKA. At that moment we only had two 1mL columns, one was HP and the other XL, and I was using those connected, and my purification worked great. My wash A buffer had a 500mM NaCl and elution was 1M NaCl. The protein has a positively charged tag so it stuck to the column really well.
However, they started having a lot of pressure issues and we’re expired and we ordered new ones but only XL. Now during the wash phase everything looks the same, but when I increase NaCl concentration there isn’t any kind of peak.
has anyone had this issue with new columns?
Did you connect two XL in serial instead of HP + XL or just use a single XL...?
0.5 M NaCl is a bit aggressive to use as a wash buffer if you prefer to use 1M NaCl as an elution buffer ...Anyway assuming you are connecting two XL instead of XL+HP, for your case it seems your POI is more retentive at SP Sepharose HP resin and less at Speharose XL resin. Definitely, this new configuration offers a novel method to be integrated and the first action should be decreasing the NaCL conc of your wash buffer. You may decide your final strategy by confirming if the POI eluted during the washing step at 2XL config.
- Badges
- Science topic
- Similar topics
- Mathematics
- Statistics
- Regression