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Will 10 min boiling disrupt BS3 cross linking?

Immunoprecipitation

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7 replies to this topic

#1 Sander

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Posted 22 December 2013 - 09:09 AM

Hi, 

 

I am using BS3 as a cross-linker for my co-immunoprecipitation experiment but I have not been succesfull so far.

I know BS3 is a non cleavable cross-linker and the associations formed are described as: "are not easily broken"

Now I am wondering if boiling my samples (95°C) for 10' in SDS sample buffer will be sufficient to brake this bond or not..

Hopefully someone can help me out here?

 

Thanks!



#2 memari

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Posted 23 December 2013 - 08:37 AM

It is Membrane-impermeable, allowing for cell surface labeling

http://www.piercenet.com/product/bs3


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#3 Sander

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Posted 23 December 2013 - 08:54 AM

Thank you for your response memari.

 

Yes, I know it is membrane-impermeable, but that has nothing to do with my question..

Will the bond, made by BS3, brake if I boil my sample with SDS for 10 minutes at 95°C?



#4 neuron

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Posted 23 December 2013 - 11:57 PM

Ideally, it should not brake, as the crosslinking is via covalent bonding, and covalent bonds are very strong. Practically also, I have used BS3 and it shows crosslinking even after boiling the protein sample. 



#5 Sander

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Posted 24 December 2013 - 12:38 AM

So I will have to use another cross-linker if I want to be able to brake this band after IP. Any suggestions of a comparable cross linker (membrane impermeable) that can be cleaved?



#6 mdfenko

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Posted 24 December 2013 - 04:37 AM

this may be what you are looking for: lomants reagent from thermo-pierce

 

i found that with google.


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#7 Sander

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Posted 24 December 2013 - 05:11 AM

Thank you for your try mdfenko, but you just suggested a membrane permeable crosslinker..

There are tons of cross linkers available from pierce, that is why I am asking here if someone knows one from practice that is comparable to BS3, but cleavable ;)



#8 neuron

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Posted 26 December 2013 - 04:08 AM

BS3 is amine to amine crosslinker, if you just fill requirements of BS3 in crosslinker selection guide you find this-

 

http://www.piercenet...selection-guide

 

In this two are cleavable, I have never used them, But you can try. DTSSP looks more closer to BS3.







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