What is the size of the column? Can the lectin bind to agarose or dextran?

If the lectin does bind to the resin, then you may be able to prevent that by including in the eluent a high concentration (like 1 M) of the sugar to which the lectin binds, to compete with the resin.

What mean no defined peak? and what did you expect? protein where purified?

which is mw and extinction coefficient at 280nm of your protein ?

500ul is the maximum volume that you can load to this coloumn. Generally I'm using 100u with the same coloumn because lower volume can improve the resolution but of course in your specific case the results may depends also from the buffer composition and aspecific binding the the resin as suggested from Adam that may result in a sort of smear.

ciao

Manuele

What running buffer did you use? What was the sample matrix?

Omar Gonzalez-Ortega Hi, Omar! the bed dimensions is 10 x 300 mm and the volume is approximate 24 mL. Lectin does not interact with agarose and dextran

Just found this: https://doi.org/10.1016/S0378-4347(00)85167-1

I cannot read the article, as I have no access, but the title sounds promising:

"Anomalous behavior of lectins in size-exlusion high-performance liquid chromatogarphy and gel electrophoresis"

Achim Recktenwald Hello Achim!

The buffer I used was 0.05 M sodium phosphate and 0.15 M NaCl pH 7.2. It is a lectin of basiodiomycete fungus called Langermannia bicolor

Manuele Martinelli Hi Manuele! I'm suspecting that if I concentrate the sample more and apply a smaller volume I might improve the chromatogram. reading at 280 nm is quite low I usually read at 214/215 nm. In Adam's response I show the resulting gel filtration chromatogram, I expected a single peak since in electrophoresis the sample was very clean and with a single band of about 112 kDa.

Do you have perhaps cross-linking or aggregates in your sample?

The peak is extremely wide for a 30mL column, almost 22mL. If your protein was mostly pure and if there is no binding to the resin, then this peak looks like you have many different species eluting.

Did you use chaotropes during your protein purification? If yes, was the protein refolded properly or do you have many differently folded versions that interact with each other?

Hi Geovanna

Maybe you should take into account the isoelectric point of your protein. If you are working close to the isoelectric point you can have different species with unbalanced charges, and this could be the explanation of the different peaks.

Increasing ionic strength of the buffer and move away from the isoelectric point could be a possible solution.

Best of luck

A lack of 280 nm absorbance could be due to a very low proportion of aromatic amino acids in the protein.

Multiple peaks could be due to aggregation of the protein. This would not show up on SDS-PAGE. Try dynamic light scattering, if an instrument is available.

maybe the height of column is not proper for separation different species from each other. try a column higher. check the viscosity of the sample, if it was too viscose, (e.g. because of HCD or other impurity) resolution of separation would be terrible.

For SEC, the general sample volume is 1% CV, or at most 2%. Your CV is 24ml, hence 500uL sample volume may negatively affect the performance. Still based on the chromatogram, there are large aggregates over 500kd in your sample. Perhaps your lectin has other conformations such as dimer or tetramer but at a lower ratio compared to monomer, therefore not quite visible on gels.

Perhaps the old Pharmacia guide to gel filtration can help you: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/General_Information/1/ge-gel-filtration.pdf

How about adding some sugar (maltose etc) in your gel filtration buffer for the isolation and buffer exchange your lectin protein sample with it prior to loading sample into sample loop for isolation on an S200 column.

Or add sugar from the very beginning such as initial purification steps (capturing stage) before gel filtration.

If you still have the same problem then perhaps trying an ion exchange chromatography will not be a bad idea - use a resource Q or S column to improve resolution of lectin species if their is any in the sample.