I'm quite new to protein production and purification and have a very basic question.
Realistically, what is the minimum that I can get away with in terms of protein sample quantity/concentration with size exclusion chromatography? I have a small sample of protein that I would like to run on a size exclusion column to check the profile (not be fractionated) so I can compare it against another sample of the same protein that I transferred into a different buffer (I have more of this sample compared to the first).
The protein sample is about 500ul and at 0.5mg/ml, so about 250ug total. I have access to Superdex 200 10/300 and 5/150 columns (as well as larger 16/600 and 26/600 ones that I assume are too big for my small amount). I have 100ul and 500ul loops available.
I'm thinking of using a 10/300 column and concentrating the sample down for loading via a 100 ul loop. Is that realistic thinking or do I simply not have enough protein for doing a size exclusion chromatography?
Thanks.
Depends on your detection system - for RI det. the conc. is too low. For light scattering detectors or UV it is ok.
For proper molecular mass distrib. determination you need RI or LSD.
Regards
G
O.K thanks, the detector is UV. I'm not too concerned about the molecular weight aspect as my protein is shaped differently to what the standards assume so it always comes off ealier than expected for it's molecular weight.
What system and what kind of UV detector are you using?
Your UV detector type will influence sensitivity, the smaller the flow cell the better the resolving power.
A 5/150 will allow you to use less sample, there is no benefit for your application (analytical chromatography) by using larger columns (preparative chromatography).
If you load your sample correctly you will get a chromatographic profile of your protein with what you have and could load 10-50ul of your unconcetrated sample.
The main problem people encounter using small loops is inadvertant entry of air into the loop and this throws your sensitivity of your experiment by giving huge air peaks in the chromatogram.
So the best way to load the sample is:
1: flush the loop with your GF buffer, use a syringe to load buffer
2: switch the loop to inject (no flow), remove the syringe used to flush
3: place your syringe with sample in the injection port
4: switch the loop to load
5: load your sample, do not remove syringe
When there is no flow and you switch the loop to inject, it goes inline with the column and system fluidics. When you remove the syringe no air can enter the loop.
Practise with some filtered BSA or GF standards.
You should get nice clean chromatograms.
Be mindful of what you are trying to analyse and how you do it.
If you use the same GF buffer for both you may get the same profile!!!
You should really analyse in the buffer in which you have your protein to see how it is behaving in that buffer.
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