If I want to use nuclei immediately for a co-IP, again should...
If I want to use nuclei immediately for a co-IP, again should I resuspend with 400 μl NSB buffer after I discard the supernatant at the end of Step 8 ?
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Fang Xu Author Answered Aug 23, 2017
Shandong University
Hi Emilio, NSB is used to store the nuclei which won't break the nuclear membrane. I use either sonicaction or sonication combined with high salt to break the membrane. I have worked with different proteins. It seems that some protein is easy to be released from Nuclei, while for some protein, it's hard. You can play with the condition. Sorry for missing your questions earlier and hope this is helpful for you.
Hi Fang,
I am using this protocol for Co-Ip experiment.. I would like to know how your break the nuclear envelope.. I have seen that you use different buffer to NSB, adding this buffer is enough to break the nuclei or I need some additional step?
Thanks
Fang Xu Author Answered Mar 30, 2015
Shandong University
Yes,suspend the pellet using the new buffer instead the NSB. How much volume depends on how you want the nuclear diluted. I usually suspend nuclear of 10g Arabidopsis in 1-2 mL buffer.
Ahmet B Answered Mar 30, 2015
University of Massachusetts Amherst
but how much volume, do you suggest to resuspend the pellet from last step?
Fang Xu Author Answered Mar 30, 2015
Shandong University
NSB buffer is used for store the nuclei as you can tell that it contains high concentration of Glycerol and Sucrose. For Co-IP experiment, you need to break the nuclear envelope and solubilize the protein for Co-IP. I use different buffer instead of NSB to suspend the nuclei for further step. Here is the recipe for your reference:20 mM HEPES-KOH pH 7.9, 2.5 mM MgCl2, 100 mM KCl, 20% (v/v) Glycerol, 0.2 mM EDTA, 0.2% Triton X-100, 1 mM DTT (add before use), Protease inhibitors (add before use), optional: Phosphatase inhibitors (add before use).
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