We have a protein (34 Kd) fussed in the N-terminal to GST and we are having problems with the binding to a GST affinity column. We tested different pH´s (6-8), buffers (Tris, PBS, phosphate), ionic strengths (0-500 mM NaCl) and DTT concentrations(0-10). We also tried at low flow rates(0.1 ml/min) and different temperatures (4-25 C) and also different sample concentrations. We tried with and without ysozime (1 mg/ml) in our binding buffer and we have tried different sonication protocols (trying not to affect GST structure).
We can cut our protein from GST with a protease without major problems.
Anyone has any other suggestions or ideas?
Many thanks!
Juan
Yes, we did test with 2 new GST affinitty columns that work well with GST controls. And, because of our protein, we can not put the GST on the C-terminal. We are also trying to purify te protein without using GST affinity columns (but we need to express our protein attached to GST because we obtain very little expression in other vectors as pMal, pET-Sumo, pET-11...).
Hi there,
Maybe an unproper folding of the GST domain... Have you tried expression at lower temperature?
Your protein of interest might also hide GSH bindind site. You might try non denaturing concentrations of detergent or urea.
Hi,
To improve binding, you could try to incubate your lysate with GSH beads for a few hours or even overnight at 4°C and pour a gravity flow column next day.
Did you sequence the GST-tag of your vector? Maybe, check for any mutations in your tag that might impair the binding efficiency.
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