I have done cross linking experiment with 0.1 % glutaraldehyde with native, reduced and oxidized form of protein (~19 Kda). In all case several bands were observed of higher molecular weight. what could be the reason behind it. Secondly I have done gel filtration which showed single peak around 75ml in superdex 20/200 column. Please do suggest me?????????????
Dear Anamika,
This is not surprising since "reduced versus oxidized forms of a protein" refers to cysteinyl residues (SH groups). Your work demonstrates that there is no reacting SH groups at the surface of your protein that could be responsible of the formation of some quaternary structures.
Glutaraldehyde reacts with lysines not with cysteines, forming Schiff base with the amine group (NH2). Your experiments shows that there are lysine residues available at the surface of your protein! The two conclusions are not mutually exclusive at all!
Best regards,
Pr Philippe Urban
Most probably your protein form oligomers resulting from non-covalent bonds (which you prove with cross linking step) , which are distroyed during the ectrophoresis, that's you see the protein as monomer on gel ..
Dear Anamika,
This is not surprising since "reduced versus oxidized forms of a protein" refers to cysteinyl residues (SH groups). Your work demonstrates that there is no reacting SH groups at the surface of your protein that could be responsible of the formation of some quaternary structures.
Glutaraldehyde reacts with lysines not with cysteines, forming Schiff base with the amine group (NH2). Your experiments shows that there are lysine residues available at the surface of your protein! The two conclusions are not mutually exclusive at all!
Best regards,
Pr Philippe Urban
Hi Anamika,
Your question can be subdivided into three questions:
1) why glutaraldehyde gives multiple bands?
2) why you get similar multiple bands under different conditions?
3) Does superdex single peak corresponds to a monomer peak?
Answers:
1) Glutaraldehyde cross linking is used to detect weak protein-protein interactions, which otherwise cannot be detected by common methods such as size exclusion chromatography. Due to their weak interactions, oligomers tend to dissociate thus giving a monomer peak in the superdex column. However, when you add glutaraldehyde, these transient interactions lead to cross-linked product which you observed as several bands in the gel.
2) This is an interesting observation: native, reduced and oxidized conditions result in similar multiple bands. It could be interpreted in several ways but in my opinion, multiple bands suggest that weak/transient interactions (Using my first point, I am concluding that your protein is undergoing transient interactions) are independent of the thiol status (oxidized or reduced). It could be due to hydrophobic interactions and requires further investigations.
3) You observed a single peak in a superdex column. You haven't mentioned how broad the peak was. Usually a polydisperse samples (such as yours with weak/ transient interactions) will have broad peak as opposed to a monodisperse samples. Therefore, under certain conditions such as yours single peak can be misleading and the result should be treated with caution.
I hope this answers your question.
Good luck!
Abhay
Hi Anamika,
I think Abhay Thakur is right, what I wanted to highlight is the appearence of similar bands under different conditions. This phenomena is "unusally common" and there is no real explanation for this. However, this often happens due to strong hydrophobic interactions (as Abhay already mentioned), which cannot be separated even by hard preparation conditions (heat, SDS, etc.). There is somewhere already a discussion about this in researchgate, but I didn´t fin it ... sorry. Furthermore, the cross-linking of proteins with glutaraldehyde is not that clear as it seems (according to literature), in some cases I use glutaraldehyde in protein crystallization experiments to make my crystals less fragile (for soaking experiments) and under some (unfortunately not really reproducible conditons) glutaraldehyde is producing polymers which I once "catched" on my reducing SDS Gel (high MW bands) .... So, I would suggest to do e.g. dynamic light scattering, this is more appropriate than superdex and will give you more hints about the state of your protein in solution.
Regards,
Aleks
Aleks
Hi again Anamika,
I did not understand one point you present ... Did the gel filtration chromatography perform or not on the glutaraldehyde-treated protein?
Philippe
Following up on above explanations, this might be a good opportunity to learn more about your protein. You might be working with a protein with quaternary structure, but the chromatography conditions you have been using have promoted dissociation of the subunits. If the mol wt of the bands that you observe are simple multiples of 19 kD, you could build a hypothesis with regard to the composition of the full protein, and test it.
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