Starting from a crude extract, it will not be possible to achieve complete purification with only a gel filtration step. Chromatographic protein purifications usually have multiple steps employing different properties of the protein.

Is your 50 kDa protein eluting at the expected volume for a protein of its size, based on standards? It may be an oligomer and therefore have a higher molecular weight , or it may be aggregated and elute in the void volume. Or, it might be degraded and elute as a mixture of smaller fragments.

All of these gel filtration methods rely on both shape and size of the protein but will remove much of the other shape and size proteins, Your next stage of purification could be ion exchange column chromatography to remove differently charged proteins, The resins will have diluted your sample so another good method for purifying diluted protein is isoelectric focussing which not only separates differently charged proteins but also concentrates your protein while it is doing it.

What makes you think that there is no purification...do you hve a specific test/activity for this protein. Sometimes enzymes need cofactors and the purification method removes the cofactor so that it looks like you are losing specific protein but actually there may be some purification happening

Thank you so much Adam and Paul. Basically, the purpose of my project is to understand how different resins affect the purification of my protein not to actually purify the protein. I did think it may be an oligomer therefore the weight that I got using SDS PAGE may not be accurate.

Paul, you mention in your post that gel filtration methods rely on both shape and size of the protein which I understand, but then you mentioned that they will remove much of the other shape and size proteins... would you mind clarifying what you mean by that please?

Thank you both again, really appreciate it !

Ok gel filtration matrices are cross linked polymers with the degree of cross linking defining a pore size within the bead rather like PAG gels.

They act as molecular sieves so large molecules cannot get into the matrix, flow round the outside and elute very quickly. Small molecules have to fight their way through all of the gel volume so elute slowly. In between we have a good separation situation where different size molecules will move at different speeds through the matrix so separate but this movement depends on both shape and size so a long rod shaped protein will move slower through a column than a tight compact spherical protein so you can get separation even of proteins of the same molecular weight because some are rod shaped,some are spheres and some just irregularly untidy on the outside ( ends sticking out. The shape and size will also depend on the acidity of the elution buffer as shape will be determined by the charge on the amino acids on the outside of the protein being solvated so larger than an equivalent protein with non polar amino acids on the outside which will not be solvated as much. In some cases with multimeric proteins held together by disulphide bonds they may become unstable when run in conditions that are oxidising and fall apart into smaller sub units

Regarding your original question if the gel excludes your protein then there will be no purification from any protein above 50kd but if the protein does run through the matrix then there will be good separation from proteins of different MWts but there will be band broadening of all proteins flowing through the column so excellent separation of close size proteins can never be achieved on gel filtration. You have to check the separation size range of each gel to determine which are including 50KDa in their separation range to see which are the most useful

The gels that i have used seem to be appropriate for a protein of 50KDa as their fractionation ranges are between 3,000 - 400,000. What I find confusing is that with the exception of two gels, protein 5 eluted in the same fractions (15 to 35) and with a similar number of remaining proteins (3 proteins) which have similar molecular weights (50KDa, 30KDa, 20KDa). The two "outlier" gels have a fractionation ranges of 6,000-70,000 and 3,000-60,000 and in their cases there are more contaminating proteins at the end (5-6) of various MWs ranging from 20 - 70KDa . I am assuming that this is because the weight of my protein (50KDa) is at the high end of these two gels' fractionation ranges therefore it was eluted in the fractions with a higher number of other proteins.

Since most of the resins you used have much the same sieving properties, it should not be surprising that they give similar purification results.

There are chemical differences in the resins which could have an effect on the separation if the protein of interest has an affinity for one resin over another, which would cause a later elution of that protein.

The resolution of the separation, i.e. the sharpness of the protein peaks, will be affected by the bead size, with higher resolution obtained with smaller bead size. This comes at the cost of higher back pressure or slower elution.

Resin types also differ in their pressure tolerances. Some are appropriate for systems that operate above atmospheric pressure, other are not.

Thank you both. This is my first time purifying proteins and your answers really helped.