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disactivated the remaining epoxy goup on the surface of microarray

peptide array

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#1 xuanyuanhu

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Posted 11 July 2013 - 07:08 PM

How to disactivate the remaining epoxy group on the surface of microarray with the small molecular in mild condition? Usually, the blocking agent was BSA, whose MW was about 68kD, could block the capture agent binding with the targets. Therefore, smaller MW of blocking agent (or disactivating agent) was vital in my experiment. Which agents was fit to my condition and chose in my condition?

#2 mdfenko

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Posted 12 July 2013 - 04:12 AM

you can try casein (alpha or beta or hydrolysate) if you're not probing for phosphoproteins.
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#3 xuanyuanhu

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Posted 12 July 2013 - 07:04 AM

you can try casein (alpha or beta or hydrolysate) if you're not probing for phosphoproteins.

According to my experimental results, the effect of casein was similar with the BSA

#4 Winegarden

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Posted 12 July 2013 - 10:49 AM

Hi Xuanyuanhu,

I assume you are using the arrays for protein work since you are blocking with BSA? With olgios many people have had success negating any left over epoxy groups using ethanolamine (which of course is much smaller than BSA) so that might work. You can also try some commercial protein free blocking buffers like Pierce's one http://www.piercenet...9-07774ABB3041.

We've had good success using SEABLOCK from Pierce as well for protein applications.

I'd start with ethanolamine though - it's cheap and quite effective.

#5 xuanyuanhu

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Posted 12 July 2013 - 05:51 PM

Hi Xuanyuanhu,

I assume you are using the arrays for protein work since you are blocking with BSA? With olgios many people have had success negating any left over epoxy groups using ethanolamine (which of course is much smaller than BSA) so that might work. You can also try some commercial protein free blocking buffers like Pierce's one http://www.piercenet...07774ABB3041.

We've had good success using SEABLOCK from Pierce as well for protein applications.

I'd start with ethanolamine though - it's cheap and quite effective.

The ethanolamine was also tested, but it did not work well. The following conditions was tested:

a.50mM ethanolamine in 0.1 M Tris buffer (pH 9.0) was tested in 37 or 50℃。
b.1M ethanolamine in ion-free water, and the pH was adjusted to 9 by 37% HCl. This mixture were also tested in 37 or 50℃。

The commercial available blocking agent, such as Nexterion Block E from the SCHOTT, did not work well, also.

Any tips or details existed in this process?

#6 Winegarden

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Posted 19 July 2013 - 09:34 AM

Hello again,

What were the problems with ethanolamine? High background?

I assume when you use BSA you get low signal but with ethanolamine you get high background?

Which epoxy slides are you using? Schott?

#7 xuanyuanhu

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Posted 21 July 2013 - 08:33 AM

Hello again,

What were the problems with ethanolamine? High background?

I assume when you use BSA you get low signal but with ethanolamine you get high background?

Which epoxy slides are you using? Schott?

As you assumed, the backgroud was high when the ethanolamine was used as the disactivating agents. Therefore, the ethanolamine was probably not working well. By the way, the epoxy slides was purchased from CapitalBio, a microarray corporation in China. The provider was immportant?

#8 Winegarden

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Posted 24 July 2013 - 11:33 AM

The supplier is not necessarily important no - but we have seen variable results with different suppliers. Not everyone does the coating the same - and some seem to work better than others.

You could try reducing the epoxide but I'm afraid that might affect your peptides. NaBH4 will reduce the epoxides to alcohols. This chemistry is often used when dealing with aldehyde surface chemistry (to reduce the imide bond made from the Schiff Base reaction).

The problem is the reaction of an amine and an epoxide in water is kind of slow apparently and you do not always full reactivity. I came across the paper that talks about it a bit and may have a solution for something else to try. Otherwise sorry - not sure I can think of what else to try....

http://www.sciencedi...2006018461?np=y

#9 xuanyuanhu

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Posted 24 July 2013 - 10:35 PM

The supplier is not necessarily important no - but we have seen variable results with different suppliers. Not everyone does the coating the same - and some seem to work better than others.

You could try reducing the epoxide but I'm afraid that might affect your peptides. NaBH4 will reduce the epoxides to alcohols. This chemistry is often used when dealing with aldehyde surface chemistry (to reduce the imide bond made from the Schiff Base reaction).

The problem is the reaction of an amine and an epoxide in water is kind of slow apparently and you do not always full reactivity. I came across the paper that talks about it a bit and may have a solution for something else to try. Otherwise sorry - not sure I can think of what else to try....

http://www.sciencedi...2006018461?np=y

Thanks very much. These method would be tested in our lab very soon.




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