Is there a way to screw up dialysis? - (May/03/2008 )
I must be missing something obvious.
I am trying to remove all of the excess fluorescent die from one of my reactions. The MW of the die is about 450 and I am using a dialysis tube with a MW cut off of 3500.
I was suspicious that not all of the free die was leaving the tube, so I set up one dialysis bag where I placed about 0.5 mg of die in 10mL of DMF and placed it in the dialysis tube. The tube is in about 5L of H2O with stirring and I have been changing the buffer about 3 to 4 times a day for the past 3 days and the solution inside the tube is still very fluorescent.
The MW of the die is well below the MW cutoff of the dialysis tube and the die is suppose to be moderately soluble in H2O. I must be doing something wrong.
One more thing that I noticed was that yesterday I opened the dialysis tube to take out a sample to check for fluorescence and after closing the bag again it has not swelled up with water. Is it possible for the pores in the tube to close and become impermeable to water after some time? Or am I completely crazy?
I assume you checked with TLC for free dye inside the bag?
Solubility and ionic interactions may account for the abnormal retention inside the bag.
Can the dye interact with the molecule you conjugate to by ionic interaction? See if you dialyse it against 1 or 2 M NaCl during the initial 24 hrs helps?
You can use organic solvent instead of water if solubility is an issue. You need to consult it with the company if the bag can be used in the solvent you choose.
The initial swelling in the first a few hours is due to DMF and water exchange. Once DMF leaks out, no further expansion would take place.
You may want to dialyze against DMF or a DMF/water mixture. I would test solubility of the dye in water. You may find it to be essentially insoluble, which would explain your difficulty.
I am trying to remove all of the excess fluorescent die from one of my reactions. The MW of the die is about 450 and I am using a dialysis tube with a MW cut off of 3500.
I was suspicious that not all of the free die was leaving the tube, so I set up one dialysis bag where I placed about 0.5 mg of die in 10mL of DMF and placed it in the dialysis tube. The tube is in about 5L of H2O with stirring and I have been changing the buffer about 3 to 4 times a day for the past 3 days and the solution inside the tube is still very fluorescent.
The MW of the die is well below the MW cutoff of the dialysis tube and the die is suppose to be moderately soluble in H2O. I must be doing something wrong.
One more thing that I noticed was that yesterday I opened the dialysis tube to take out a sample to check for fluorescence and after closing the bag again it has not swelled up with water. Is it possible for the pores in the tube to close and become impermeable to water after some time? Or am I completely crazy?
In my latest attempt I wanted to see if the die could exit the membrane so I only have the die in the bag and nothing for in to conjugate to.
I will check the solubility of the die in H2O again and I may try an organic solvent. Thanks
I checked the solubility in water and it isn't great, but it does go into solution. What are some inexpensive and non toxic or less toxic solvents I should try?
try ethanol first.
can you use gel filtration to separate free dye from proteins?
I was planning on giving GPC a try, someone recommended that I use these beads:
(Sephadex G-25, 20-80 um, molecular weight cutoff of 5000; eluent: 20%
methanol in water).
Does anyone have any favorite protocol for GPC, what kind of column do I load the beads into and where can I buy them?
(Sephadex G-25, 20-80 um, molecular weight cutoff of 5000; eluent: 20%
methanol in water).
Does anyone have any favorite protocol for GPC, what kind of column do I load the beads into and where can I buy them?
tell us whats the component that will be labeled. PD-10 (G25) columns from GE healthcare is very easy to use if the component is >3000 Da and it has an instruction comes with the product.