I have a complex of 7 proteins that I am recombinantly expressing and purifying on nickel (1 of the proteins has a his tag). I know by coomassie staining and mass spec that all 7 proteins are present. These proteins should form a ring structure, but when I look by EM I am not seeing the rings. I want to try crosslinking the proteins to each other as soon as they come off of the nickel column in case the ring is very unstable. What is the easiest/best protocol for protein crosslinking? Formaldehyde, glutaraldehyde, DMP?
Hi Martha, if your complex is stable at ~pH 6, then I would recommend using the zero-length EDC crosslinker.
If it is only stable at pH over 7, then formaldehyde would probably be the way to go.
My favorite crosslinker is BS3. It will crosslink amino groups and has a spacer. Most importantly, it is water soluble.
There are some things to keep in mind. (1) The buffer in which amine-to-amine crosslinking is done should not have any primary amines. Sodium bicarbonate (0.1M) is recommended. (2) You should experiment with different crosslinker combinations to find the minimal concentration that gets the job done. Using more can result in a lot of artifacts. (3) One crosslinker may not work for all the proteins if they do not have suitably located residues.
I have used BS3 before for crosslinking antibodies to Dynabeads. The protocol says to use 20-50 fold Molar excess of the crosslinker if the protein concentration is
I would be concerned about the high concentration of imidazole. Can you remove it by dialysis?
I can remove it, but I would like to prevent any manipulation of the the complex before cross linking. It has to come off of the nickel as a complex of 7, as only 1 protein has the his tag. I am worried that a lot of it may become dissociated during dialysis. Imidazole has secondary amines, not primary, I think. Does anyone know if cross linkers that are inhibited by primary amines would also be affected by secondary amines?
Hi Martha, imidazole has at best a secondary amine with reactivities between pyridine and pyrrole:
https://www.researchgate.net/profile/Arlette_Richaud/publication/49786909_Chemical_Reactivity_of_the_Imidazole_A_Semblance_of_Pyridine_and_Pyrrole/links/00b4953c57a0fe7c92000000/Chemical-Reactivity-of-the-Imidazole-A-Semblance-of-Pyridine-and-Pyrrole.pdf
Most amine reactive crosslinkers are very ineffective at linking secondary amines. So I would think that you are safe using primary amine crosslinkers in the presence of imidazole.
Article Chemical Reactivity of the Imidazole: A Semblance of Pyridin...
Yep, imidazole should not be a problem. I would use glutaraldehyde, which is cheaper but it depends on your final application.
I would prefer you use glutaraldehyde being a relatively cheaper crosslinker and easy to work with.
Does this protocol sound reasonable? 50-100ug proteins in 100ul + 5ul 2.3% glutaraldehyde for 2-5min at 37C. Quench with 10ul 1M Tris-HCl, pH 8.0
It could be, it depends on the number of lysine residues on the surface of your proteins. I prefer to run reactions with glutaraldehyde and proteins at 4C for 2 h. At 37 C the reaction rate will be fast and since you cannot vigorously stir your protein sample you need to give glutaraldehyde some time to generate homogeneous reaction conditions. Add a reduction step using sodium borohydride.
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