We are trying to purify novel lectin which presence is shown by the hemagglutination tests. But first of all we need to know the affinity to carbohydrates of potent lectin. Any Ideas?
Hi Gabriele, you could try competition binding studies using labeled lectins from Vector labs.
https://www.vectorlabs.com/catalog.aspx?catID=59
Lectins are proteins that recognize and bind specific carbohydrates found on the surfaces of cells. They play a role in interactions and communication between cells typically for recognition. Carbohydrates on the surface of one cell bind to the binding sites of lectins on the surface of another cell. Binding results from numerous weak interactions which come together to form a strong attraction. A lectin usually contains two or more binding sites for carbohydrate units. In addition, the carbohydrate-binding specificity of a certain lectin is determined by the amino acid residues that bind the carbohydrate. Lectins are specific carbohydrate-binding proteins.Rapid binding kinetics also facilitate the binding of lectins to carbohydrates. Mainly binding of sialyl Lewisx (a tetrasaccharide) to P-selectin. Rapid binding kinetics allows for spatial complementarity to be reached between a low-energy conformation of the carbohydrate and the prearranged binding site of the lectin. The shape of the binding sites in carbohydrates plays a factor in its bondage to lectins. An example of this is the case of galectin-1 binding to ganglioside GM1 (a pentasaccharide).
Hi Ravi, why are you doing a copy/paste from wiki and not acknowledging the source ?
http://en.wikibooks.org/wiki/Structural_Biochemistry/Carbohydrates/Lectins
"Lectins are proteins that recognize and bind specific carbohydrates found on the surfaces of cells. They play a role in interactions and communication between cells typically for recognition. Carbohydrates on the surface of one cell bind to the binding sites of lectins on the surface of another cell. Binding results from numerous weak interactions which come together to form a strong attraction. A lectin usually contains two or more binding sites for carbohydrate units. In addition, the carbohydrate-binding specificity of a certain lectin is determined by the amino acid residues that bind the carbohydrate. Lectins are specific carbohydrate-binding proteins."
A straightforward way of measuring lectin-carbohydrate binding affinity is isothermal titration calorimetry, but many other techniques can be employed for studying binding interactions.
See, for example: https://www.researchgate.net/post/What_is_the_alternative_method_to_study_ligand_receptor_binding_experiment_beyond_radiolabelling
Thank you all so much for your informative and useful answers!
If I understood right, these methods are used for purified lectins. My case is different. I have whole bouquet of proteins extracted and I need to detect lectins in it. I think that the best way would be detect lectin carbohydrate specificity and then purify in affinity chromatography column. What would you suggest?
Thank you so much again for the answers!
Hi Gabriele, OK, are you looking to identify specific lectins in your prep., or are you looking to identify all lectins in your prep?
I would like to identify all lectins in my prep. My lectins are plant origin.
OK, identifying all lectins in your prep. is a lot of work.
Consider these papers:
http://www.jbc.org/content/252/1/57.full.pdf
http://cdn.intechopen.com/pdfs-wm/33048.pdf
Alternatively, since you are looking at hemagglutination, you could fix RBCs, and identify lectins that bind to them.
http://www.sciencedirect.com/science/article/pii/0161589079900026
Thank you so much, Steingrimur! Those paper is very useful! :)