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Dual cross-linking thoughts


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16 replies to this topic

#16 chipy

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Posted 23 July 2012 - 10:18 AM

Radish,

I am not using agent mentioned above. I overexpressed the proteins and I got the results! I would like to try using the agent now...could you please let me know the protocol? Thanks

For the question you asked, I have no idea whether we can western blot xlink samples. what is your approach? You harvest the samples, do IPs follow by western?


Hey Giny

I use the upstate protocol, so the only thing I've changed was the crosslinking.
I used attached cells (arround 2x107 cells in a 100mm plate) washed them with PBS+protease inhibitors 3 times, added the agents with 10 ml of PBS=PI (the agents were all solubilized in DMSO in 1 M stocks and added to the final concentration of 1,5 mM) incubated for 1 hour with agitation at room temp, washed again 3 times with PBS, added 10 ml of PBS+PI and 1 % Fa and incubated for 10 minutes. The rest of the protocol is the same, add the glycine yada yada.

The 1.5 mM concentration on DMSO or PBS? I see it precipitates in PBS!!!

#17 guru

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Posted 22 July 2014 - 11:55 AM

DSG seems to provide the best results.  Using ProteoChem's DSG (http://www.proteoche...100mg-p-45.html) in conjunction with formaldehyde has proven to be very effective (Nowak, D.E. et al 2005, BioTechniques 39:715-725).






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