Concentrating protein - (Jun/27/2008 )
we have GCSF protein produced in inclusion bodies. we solubilise it refold
it and then concentrate it using proflux system. still the concentration
of the protein we get is less and volume is more for us to load in Ion
exchange chromatography. is there any simple technique that can be
performed in lab without using any hi-fi instrument to concentrate the
protein in small volume of buffer. the protein we obtain is about 0.7
mg/ml and we get 450 ml solution. our column is 110ml lab scale and we
have to work in that only. i have heard of NH4SO4 precipitation. but it
requires protein conc more than 1 mg/ml.
kindly help me to progress in my experiment.
it and then concentrate it using proflux system. still the concentration
of the protein we get is less and volume is more for us to load in Ion
exchange chromatography. is there any simple technique that can be
performed in lab without using any hi-fi instrument to concentrate the
protein in small volume of buffer. the protein we obtain is about 0.7
mg/ml and we get 450 ml solution. our column is 110ml lab scale and we
have to work in that only. i have heard of NH4SO4 precipitation. but it
requires protein conc more than 1 mg/ml.
kindly help me to progress in my experiment.
Ultrafiltration by centrifuge units with right MWCO will do. You can get 15 ml capacity units from millipore or several other manufacturers.
What is the salt concentration? IEC column allows you to load diluted samples and concentrate it with a step elution manner. Can you simply dilute your sample and load it on to your IEC column?
you can use a stirred cell for ultrafiltration. volume up to 400ml (more with a reservoir).
Hi biotef,
you can use ultrafiltration concentrators (100 ml volume with double concentrator unit in the same concentrators) by centrufigation. see millipore...
it and then concentrate it using proflux system. still the concentration
of the protein we get is less and volume is more for us to load in Ion
exchange chromatography. is there any simple technique that can be
performed in lab without using any hi-fi instrument to concentrate the
protein in small volume of buffer. the protein we obtain is about 0.7
mg/ml and we get 450 ml solution. our column is 110ml lab scale and we
have to work in that only. i have heard of NH4SO4 precipitation. but it
requires protein conc more than 1 mg/ml.
kindly help me to progress in my experiment.
There shouldn't be a problem loading 450 ml onto a 110 ml ion exchange column. What is the max capacity of the column? What is the max flowrate of your pumps, and the max pressure of the system? Even if you have to load slowly overnight, you can still load that much, because loading ion exchange is not like loading a gel filtration column.
thanks for your reply 
. my resin is source 15s cation exchange resin in fineline 35 column (capcity how can i find out) and max flow rate of pump is 20 ml/min but we do our experiment at 5 ml/min. and i have a probelm of high molecular weight contaminant my protein is 20 kDa and contaminant are 36 KDA and upwards which i have to separate but it does not happen i get some high mol wt contaminant even after ion exchange. how can i get rid of the same. 