Does the back-pressure increase after injecting the protein or right after you equilibrate your column with the mobile phase? Do you know if your protein is stable in the mobile phase you are using? Have you done a blank run after the wash step to make sure the column does not have any remanent protein or ligand?

A possible problem is that the small molecule ligand is not soluble in the mobile phase at the concentration you are using, causing the column to clog, leading to high back pressure and irreproducible results. You might be able to solve this problem by reducing the ligand concentration, or changing the mobile phase to make the ligand more soluble (change pH, lower ionic strength).

thank you Cesar and Adam, in fact the back-pressure elevation is not immediate it developed with multiple injection, and my sample is well dissolved in the mobile phase, i am using only millimolar of ligand concentration and also protein, but i want to ask you Cesar what do you mean by blank run? do you mean the mobile phase which is free of ligand to clear the coloumn, i have my dout now that the problem arises from small mwt ligand since protein will be illuted and exclusded first, does it sound since, i hope so ..:)

Hey Soha!

What I mean with a blank run is to just run mobile phase and check if there is any remanent particulate in your column and if the back-pressure goes back to the typical value expected for your column. It seems that the protein concentration or the interaction with the ligand you are using causes some aggregation or becomes insoluble, and you first need to check that this is not an effect that builds up in your column until it ruins it completely. Do you know if the protein concentration you are using is within range of the maximum concentrations allowed for the column you are using? If I remember correctly, the Hummel Dreyer uses a fixed concentration of ligand and several protein concentrations to determine the binding constants, but you always have to make sure you are not overloading protein into your column.

And finally, I totally agree with Adam, maybe your ligand is not as soluble as you expect in the conditions you are using. I would equilibrate with mobile phase without ligand first and then re-equilibrate using mobile phase with ligand and check if the back-pressure built up or not, or find another way to check its solubility. You can always do your runs with a lower flux rate, but that is not going to avoid your column to clog frequently.

Hope that helps a little (I know is not that much)

Hi Soha! Is your protein an enzyme? If it is an enzyme, maybe you should also consider the solubility of your enzymatic product in mobile phase. Sometimes the solubility of product is very different from its substrate.

thanks Cesar, in fact you help me alot; i will try equilbriating my coloumn with mobile phase free of ligand, then equilbriate with the ligand, Hsi-Ho my protein is carbonic anhydrase III, it is about 30 KDa;it is water soluble,  how could i know if the product is water soluble or not; i am using 10% ACN in 25 mmolar phosphat buffer, i inject the enzyme in different concentration, but the backpressure increases not from a single run but after let me say 4 continous months????

Hey Soha,

Maybe you should check if you are recovering all your protein after size exclusioning, perhaps some of your protein is aggregating during the runs and you are not recovering all of it. For example, measure the amount of protein you are injecting and then collect your protein after size exclusioning and check if the resulting amount of protein matches the initial quantities. If not, that means your protein may be aggregating during your runs, thus clogging your column

Usually, columns like the Superdex S75 have a filter on the top side of the column (or inlet) that gets clogged with particulate matter after extensive use, specially when you are injecting high protein concentrations. You can order spare parts of this filter and change it quite easily, and in principle this should be also doable with other types of columns.

Finally, do you have a good protocol for cleaning your column in case of clogging? Cleaning your column periodically should also improve the reproducibility of your runs, specially if you are using high protein and ligand concentrations.

Hope that helps!

Thanks again Cesar for ypur concern; in fact i am not recovering the protein; instead i study the interaction of protein with ligand; which saturated the coloumn and became as stationary phase, my protein bind with ligand, i observe a negative peak then, to calculate Ki, my work is not preparative.

Hi Soha,

What i'm trying to suggest goes beyond the Hummel-Dreyer method. I'm suggesting that, ideally, whatever you load into your column should come out in the same quantity. Then, if you load X amount of protein to do the Hummel-Dryer, the same X amount of protein should elute from the column, since both your ligand and your protein are being flushed by the mobile phase (unless there is some important part of your experiment that I'm not following). But if the amount of protein that elutes after binding occurred, or even in the absence of ligand in the mobile phase, is less than what you injected, then it is your protein that is somehow getting stuck in your column and clogging it. Now, have you ever run your protein without ligands in the mobile phase and check i) if it elutes at the expected retention time for a protein of size 30 kDa? ii) if you get the same amount of protein from the elution as you injected first?.

I truly hope that your experiment works out fine!

Hi Soha! I think Cesar's suugestion is good. You can calculate the amount of protein flow in and out of column by using BCA method or SDS-PAGE. But remember to consider the large dilution factor in gel-filtration column.That means maybe you have to concentrate protein fractions flow out of column then you can see clear protein signal in your protein quantitative method. Sometimes protein will form precipitate in column because the disulfide bonds fromation, in this case you can add some dithiothreitol or mercaptoethanol in your elution buffer to avoid protein precipitate formation. Also I notice there is a metal ion inside your protein, so I suggest you to check the relation between metal ion and protein solubility. If metal ion lose will decrease the solubilty of protein, maybe you can also add metal ion inside column elution buffer. Your enzyme seems add/delete a hydroxyl group on substrate/ligand, so it will affect the solubility of substrate/ligand in aqueous buffer. By the way, the reaction of your enzyme seems envolve the proton generation/consuming, so the local pH change may also affect the solubility of protein/ligand. In this case maybe enhance the concentration of buffer can work.