Protein concentration problem ! - (Jun/05/2007 )
QUOTE (mdfenko @ Jun 7 2007, 03:48 AM)
QUOTE (baalkor @ Jun 6 2007, 11:49 AM)
Excuse me, i made a mistake. on tubes it's indicated 0.5mg. and in preparations instructions it's indicated : "the product is soluble in water (0.5mg/ml), yielding a clear to slightly hazy solution"
it says that the protein is only soluble to 0.5 mg/ml. you tried to concentrate it to ~2.5 mg/ml. it probably precipitated and is coating the membrane (unless you rinsed it, then it is gone).
did you really need to make it more concentrated than they recommended?
Yep, I would second that. If they said that the solubility of this protein in H20 is 0.5mg/ml, then that should be the max concentration you should make in H20. Unless they said anything else about better solubility in other solvents, I don't think you should concentrate the protein. Check the membrane to see if you are lucky, but next time it probably would be a good idea to be careful and read the data sheet first and follow their instruction.
By the way, in case that this protein can be solubilized at higher conc than 0.5mg/ml, or later with any other compound that you need to similarly resuspend, make solution... if they are in lyophillized form, you can easily resuspend them at the stock concentration that you want, so that there is not necessary for the following concentrating using centricon, microcon... You already got the amount of protein in the tube (very concentrated, in powdered form) and only need to add in the correct amount of solvent to get the desired concentration. You won't lose protein through additional concentrating process this way. It is easier and less time consuming too.
-Almasy-
Maybe you have to use so calle low-binding membranes. I think they are little bit more expensive, but I think it would be profitabel. I use them to concentate e.g. antibodies I produce.
(see: http://www.millipore.com/publications.nsf/docs/pf1401en00)
good luck
-moljul-
QUOTE (moljul @ Jun 7 2007, 11:04 AM)
Maybe you have to use so calle low-binding membranes. I think they are little bit more expensive, but I think it would be profitabel. I use them to concentate e.g. antibodies I produce.
(see: http://www.millipore.com/publications.nsf/docs/pf1401en00)
good luck
(see: http://www.millipore.com/publications.nsf/docs/pf1401en00)
good luck
thanks, but the links doesn't work
-baalkor-
QUOTE (baalkor @ Jun 7 2007, 01:37 PM)
QUOTE (moljul @ Jun 7 2007, 11:04 AM)
Maybe you have to use so calle low-binding membranes. I think they are little bit more expensive, but I think it would be profitabel. I use them to concentate e.g. antibodies I produce.
(see: http://www.millipore.com/publications.nsf/docs/pf1401en00)
good luck
(see: http://www.millipore.com/publications.nsf/docs/pf1401en00)
good luck
thanks, but the links doesn't work
Im sorry, I don´t know the reason why it doesn´t work! (Information: Ultracel PLC Membranes, Data Sheet - PF1401EN00, The membrane of choice for ultra-low protein binding and robust process performance) Search for the indicated product at the millipore homepage! You´ll find it.
-moljul-
QUOTE (Almasy @ Jun 7 2007, 06:23 AM)
Yep, I would second that. If they said that the solubility of this protein in H20 is 0.5mg/ml, then that should be the max concentration you should make in H20. Unless they said anything else about better solubility in other solvents, I don't think you should concentrate the protein. Check the membrane to see if you are lucky, but next time it probably would be a good idea to be careful and read the data sheet first and follow their instruction.
By the way, in case that this protein can be solubilized at higher conc than 0.5mg/ml, or later with any other compound that you need to similarly resuspend, make solution... if they are in lyophillized form, you can easily resuspend them at the stock concentration that you want, so that there is not necessary for the following concentrating using centricon, microcon... You already got the amount of protein in the tube (very concentrated, in powdered form) and only need to add in the correct amount of solvent to get the desired concentration. You won't lose protein through additional concentrating process this way. It is easier and less time consuming too.
By the way, in case that this protein can be solubilized at higher conc than 0.5mg/ml, or later with any other compound that you need to similarly resuspend, make solution... if they are in lyophillized form, you can easily resuspend them at the stock concentration that you want, so that there is not necessary for the following concentrating using centricon, microcon... You already got the amount of protein in the tube (very concentrated, in powdered form) and only need to add in the correct amount of solvent to get the desired concentration. You won't lose protein through additional concentrating process this way. It is easier and less time consuming too.
Well said. This indeed sounds like a solubility problem, and it would seem unwise/impossible to exceed the limit. If for some reason a highly concentrated stock solution is needed, check whether there're any solubility data in the Merck manual (or the data sheet for the product) and simply resuspend the lyophilized protein in the desired volume/concentration using an appropriate solvent. One cannot overcome a limit in solubility with any method of concentration. The protein will precipitate.
-Rob70-