[lee101]

Hi,
I am performing a EDC/NHS coupling reaction between my virus capsid proteins and homobifunctional diaminolinker. I am performing a two-step reaction then add the product to another preactivated capsid protein.  Since I have 3 amino group per monomers and total of 240 monomers. I have 720 accessible site for chemical crosslinking per capsid protein. Most of the time the standard protocol allow 10 molar access. My questions are as follows;

1) my protein concentration is at most 2mg/ml to prevent aggregation(most protocol work with 10mg/ml). Can I work with that concentration? cause I did not get high efficient crosslinking in my experiment based on sds reducing gel and sucrose gradient. My volume is at 100uL per protein sample.
2) can EDC/NHS be added at 720 molar excess. will it cause aggregation and how can I check that?
3) using 50mM HEPES, 500mM NaCl @pH6 and 7.5 for activation and coupling respectively. will the coupling efficiency be high?
4) I have added BME at 10 molar excess of EDC. Can I not filter it out before addition of diamino linker. Will it affect the efficiency?
My diamino linker that I added is at same concentration as EDC. Is that sufficient or too much and will aggregate?
5) for buffer exchange, I have used millipore 100kDa centrifuge to separate capsid protein(2x at 6000rpm@6mins) and excess BME/EDC. for removal of excess linker I have used dialysis as millipore method result in huge loss of protein.
5) I have tried the above protocol but end up with very little crosslinking compared to expected based on sucrose gradient. What is wrong with my protocol? Can someone help?

[magician4]

though I don't know little about the fines of biochemistry involved here , first thing that comes to mind is kinetics: if the reaction with your crosslinker follows third order kinetics - i.e. the concentrations of components A , B to be crosslinked and the crosslinker C would act like this:

v = k * [A] * [B ] * [C]

(in case of A = B hence v = k * [A]²*[C] )

it follows that,  if you reduce [A], [B ] and [C] by factor of 5 each, you'd need 5³ = 125 times more time to achieve the same result , compared to undiluted reaction kinetics.
30 minutes at 10 mg/mL  2.6 days at 2 mg/mL

just ballpark considerations, but in general, something like that might happen and turn out to be a problem, if you didn't allow for much longer reaction time than in your original protocol


regards

Ingo

[Yggdrasil]

Are you trying to get intramolecular crosslinking between monomers in the same viral capsid or intermolecular crosslinking between monomers of different virus capsids?

In either case, I'm not sure why you need to quench with βME.  It seems like you would just add EDC together with your diamino crosslinker to the virus, and react.

[Babcock_Hall]

A long time ago in an enzyme immobilization experiment I used Tris as the quenching agent, because it was a primary amine.  Were you trying to use the alcohol or the thiol end of 2-mercaptoethanol?