Dear,
I am doing in gel filtration. My protein is fgf2. In the first step of purification, I applied cation exchange chromatography, but it was not clear enough. I try to do the next step to get higher purity, by gel filtration. But every time the target protein get out of the column, it made the system over pressure.
The protein concentration of the sample is ~1.1mg/ml, the volume of sample is 500 ul. The flow rate is 0.5 ml/min. I use the Superdex 75 column of GE and the AKTA Prime system.
Thank you.
Best regards.
I also usually got pressure increase when the proteins eluted. What is the recommended flowrate for your column? Try to purify it (for me worked the best loading pepsin and incubating for awhile) to decreese the background backpressure, so it doesn't switch off, when your proteins elute.
check the flow rate of column secondly try to purify it , 15 to 20 mts incubate protein sample while before loading
What do you mean by "not clear enough"? If the sample is turbid, you will get fouling of the column. A quick spin in an Eppendorf centrifuge should remove the turbidity, if that is your protein, try to solubilise with DTT or urea. If you have to use detergent, you need to equilibrate the gel for a day or two with the running buffer because of the micellar nature of detergent solutions.
Sample volume for SEC should be about 0.5-1% of gel volume, linear flow rate should be less than 5 cm/h. GE gives away a booklet "Gel filtration: Principles and Methods" (https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/General_Information/1/ge-gel-filtration.pdf) that is very useful.
An easy solution to your problem would be to use a heparin column. Fibroblast growth factor 2 (like most FGFs) has a strong and specific affinity for heparin, and heparin columns are relatively cheap (e.g. from GE, either pre-packed or beads). Eluting FGF2 from the heparin column usually requires quite a high concentration of NaCl (probably 2 M will be necessary), and this should remove all contaminants very efficiently.
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