Home-made Size-exclusion Columns? - (Sep/23/2010 )

Hi all,

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.

Thanks,

M

Not to be greedy, but one more thing: Anybody have concerns that the approach I describe will ruin my chances of being quantitative with my assay of the 100 kDa protein's concentration?