Hi there,

The dimension of your column sounds small. Usually preparative columns have at least 120mL bed volume (see GE catalog) for a loading volume up to 5mL. So depending on the amount of protein you intend to inject I would suggest to consider the size issue because size does really matter for SEC...

About your column, it's hard to state on the volume of elution : it will depend on the oligomerization state of your protein (is it monomeric, dimeric, oligomeric?) and calibration is required for accurate calculation...

about 15 ml?

Hi there,

The dimension of your column sounds small. Usually preparative columns have at least 120mL bed volume (see GE catalog) for a loading volume up to 5mL. So depending on the amount of protein you intend to inject I would suggest to consider the size issue because size does really matter for SEC...

About your column, it's hard to state on the volume of elution : it will depend on the oligomerization state of your protein (is it monomeric, dimeric, oligomeric?) and calibration is required for accurate calculation...

I agree with Dominique and will add the following details.  Column size matters and the sample concentration and volume matters.  Measure theoretical plates then calibrate your column with the GE Gel filtration standards.  I'd set up a standard curve as follows: Choose 3 sizing standards from the kit.   -  Ovalbumin (~44 kD) is close to your protein, then chose one standard ~150% smaller than your target and one standard ~150% larger than your target - this will make a good standard curve for your target protein.  Based upon the gel pattern, there will be other proteins co-eluting with your target.

Hope this helps - good luck Best Regards

Your experiment is going to fail. You have a lot of proteins to resolve. If you still want to proceed with this fractionation add the sample volume equivalent to 0.2% of your column volume. Look up for the distributiion coefficients of the stationary phase to predict the elution volume.

To get the best possible separation your sample volume on a 24 ml column must be at at no more than 1.2 mls. This is 5% of your total volume. Also the Void volume should then be about  7 mls , and your 55kDa Protein should resolve within before 12 mls or 1/2 column volume. 

Usually you need to makes compromises with SEC. The ratio of sample size to column volume should be as low as possible, but you cannot have a column of infinite size. Over many years I settled on a sample size that is 1% of column volume as a good starting position. However, if you plan later on to have samples at much greater volume you really need to consider the potential impact of that now.

If the only purification technique that you have used so far is NiNTA, I do not think you should be using SEC as the second purification step. I would always place ion-exchange and hydrophobic interaction far ahead of SEC, unless the protein of interest is unusually large or small. In your case you have a mid-range protein (unless it is an oligomer) and SEC will most likely give a disappointing result.

I agree with Nick, try an alternative chromatographic step. I suggest to use a multimodal column. Then you combine charge and hydrophobicity.

You enzyme is not pure. Try an ionic and or cat ionic chromatography before gel filtration. Use your enzyme assay to find your fractions after chromatography

Dear All

Season’s greatings

I really appreciate your guidance.

# Ziguo Zhang Thanks!

# Dominique Liger and Dan Weaver

The enzyme of intrest is a monomer . Sure I am trying to increase the column size with column volume 100mL in L X ID 600mm x 16mm column. I will also try to use BSA (66.5kDA) and myoglobin (17.5kDa) as standards.

# Omar Gonzalez-Ortega and Vernon L Reisenbichler

Yes I already have planned to salt out protein and redissolve it into buffer so that the sample volume will only be 0.5mL.

# Nick Gee, Leif Bülow and Kaliappanadar Nellaiappan

Sure I did have a plan to use DEAE sepharose column after gel filtration. But I need to load selective fractions on DEAE sepharose.

The eluate form DEAE sepharose will then be dialysed in storage buffer.

Thank you all !

Faraz

I would also advise to try an additional ionic chromatographic step. Nonetheless, I would say go ahead with the SEC since behavior is usually predictable, but not always. If the sample can be reproduced easily, do the SEC and it will give you information on how the protein behaves.

a 120 mL column might be an advantage for larger samples, but the HR columns usually perform quite well even as preparative column. Since you packed it yourself (which is not really doable, since as far as I know the needed equipment is not available), I would think, that this might not be the case. Calculate performance parameters in water using 1%Aceton+1M NaCl as sample.

For future reference, buffer composition might influence the behavior on a SEC column, if you are exchanging the buffer during a SEC run.

Hi Faraz, I still wonder if the sequence of your columns is optimal. I see no advantage in putting SEC before DEAE, or why you would need to load selective fractions from SEC on DEAE. A potential advantage of NiNTA > DEAE* > SEC is that you can run SEC in your column storage buffer (assuming it is inexpensive), thus eliminating your proposed dialysis step. Another potential advantage of putting SEC last is that you might find that it is not actually needed, which would be a bonus if you plan to scale up later on. (*Nothing wrong with DEAE but Q or S would have a more definite charge at all pH values). Hope this helps and good luck with whatever approach your decide to use.