Microcentrifuge Gel Filtration Question - (Sep/23/2010 )
I have not found a tremendous amount of discussion on this forum about the application of gel filtration (a.k.a. size-exclusion chromatography) for small volumes.
Here is what I'm up against:
I am developing an assay for a 100kDa protein, but there is a 25 kDa non-target protein that interferes with my assay. I deal with hundreds of samples at small volumes 50-150 uL generally.
To get rid of the 25 kDa pest I am considering making my own home-made size-exclusion columns. I don't think I can afford pre-made tubes or columns/plates, they are never <$1/sample which is just way out of my budget! I guess I want to see if anyone else has done this and what their experiences were. Any advice would be helpful.
Here is the basic idea:
1. Poke a small hole in the bottom of a 0.5 mL microcentrifuge tube.
2. Put a tuft of glass wool in the hole.
3. Place the 0.5 mL tube into a 1.5 mL tube.
4. Apply a gel (Sephadex G75?) to the tube and centrifuge at low speed for a few minutes to pack.
5. Transfer the 0.5 mL tube to a fresh 1.5 mL tube to collect sample.
6. Apply sample to 0.5 mL tube
7. Centrifuge at low speed for another few minutes.
8. Store sample that flowed through in the 1.5 mL tube.
I've seen this discussed in forums here and there, but as you can see many details are missing. This is not my primary field and I've never worked with Sephadex or other filtration gels before. So here are some questions, hopefully I can fill in some of the blanks...
What gel volume should go in the 0.5 mL tube to effectively separate the 25 kDa from the 100 kDa protein?
Will the volume of gel slurry that I put in the small tube compress during packing? Will that leave room to apply sample to the top of the 0.5 mL tube? How much sample?
Is Sephadex G75 the right choice for my application?
Are there more standardized ways of plugging the hole in the base of the 0.5 mL tube? A "tuft" of glass wool is pretty vague. I've heard mention of 25 uL of glass beads... what size glass beads? What kind? Where can I find them? How do I apply "25 uL" of beads?
Sorry if these are naive questions. Any advice would be tremendous.
I would definitely be doing this in a plate. There are filter bottom plates (Millipore e.g.) that would hold G-75. Either vacuum or a plate centrifuge would pack the oolumn and pull your samples through. The technique you describe would be a good way to determine if it will work for your samples. But 100s of small tubes are not whst you want.
Hola, I donīt know the plates with filters with sephadex, by knowing how is the separation in a colum, I imagine difficulties for clearly resolve both proteins with such methods. If I were yuo I would use the microcon devices for minifugues of Millipore , it isnīt expensive and selecting a 30 or 50Kd membrane you could separate both proteins with the precaution of avoid that a little volume rest on the filter to rescue the bigest one.In other words folowing the instructions, make the first run untill only a part of volume goes througth the filter, add a similar volume of buffer upside , recentrifuge, add other volume, and repeat untill you have the 25Kd diluted in the tube and the big one concentrated in the filter, but as i say, is important make some washses avoiding that the filter get dry because the recovery of the 100Kd one coul be difficult. Good luck
Hi, I not sure what is your budget. But I do believe that buying sephadex gel is much more expensive than buy a Vivaspin protein concentrator column. For your case, you might try to use a 50K Molecular weight cut off to get rid of your 25k protein. And I do believe if is the same sample that you are using, it can be re-use for few times.
i second what AK says... that is the best bet... i have used the cut off filters atleast 10 times with only a marginal loss on the membrane (obviously for the same protein or sample)