I would like to incubate some collagen with glucose/fructose for some days. Then I would like to use the collagen for my cell culture experiments. How can I separate this collagen? Please help.
Any method to remove small molecules from large, such as gel filtration (desalting columns) or dialysis ought to suffice.
Depending on the concentration of collagen I, the solution may be viscous and the process may take some time. If your collagen and glucose incubation for several days is performed at neutral pH, you may want to change the pH to acidic range to ensure that collagen remains in solution, especially if the concentration of collagen is high (>1mg/ml). Acetic acid at 10-20mM works well. After removal of glucose, acetic acid can be neutralized as you dilute in into the medium with adequate buffering capacity in the subsequent step.
Oh! Thats very helpful information for me. Will you please give me the details?
The process is rather simple and straightforward. I am not sure what sort of additional details you need.
Oh, thanks a lot. Can I use centrifugation/filtration by filter cartridge?
"Can I use centrifugation/filtration by filter cartridge?"
Yes or no.
It all depends on exactly what you have (volume/concentration), given that all the information you have provided is rather nebulous:
"I would like to incubate some collagen with glucose/fructose for some days."
Mind-reading is a nearly extinct art. ;)
I would only recommend dialysis against 20 mM acetic acid. Collagen type I cannot be gel filtered at almost any concentration. I do agree with Tausif about the pH must be kept around 2 to complete the collagen from gelling.It can gel at concentrations as low as 100ug/ml at a neutral pH. Also only perform the dialysis and the addition of the glucose/fructose at 4C since collagen polymerization is temperature sensitive.
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