Hello everyone
I have a lysis buffer protocol that works well to detect a membrane protein (see composition below), but since my samples are precious, I wonder if I could use it also to detect a nuclear protein in the same sample? I can increase triton up to 1%, but there is no NaCl while I see that there is a high NaCl concentration in most nuclear lysis buffer.
So if anyone can tell me if this buffer could be strong enough to release nuclear proteins, or if it may work if I add NaCl, I would appreciate it ! Thanks a lot
Here is the buffer recipy:
20 mM Tris HCl (pH 7.0)
0.27M sucrose (=92g/L)
0.5% (volume/volume) Triton X-100
1 mM dithiothreitol
1 mM EDTA
1 mM EGTA pH8
1 mM Na3VO4
25 µg/ml leupeptin
1 µg/ml pepstatin A.
Hi Anne, if you are looking for DNA bound proteins, you could add DNAse to the extract and use a small bore syringe to shear the DNA with repeated uptake and dispel. (~5 times should do the trick).
I don't know if DNAse is inhibited by EDTA/EGTA/DTT, but you could add them later after the DNA has been hydrolyzed.
@ Steingrimur Stefansson
I agree with the suggestions
There is some information regarding DNAse I activity here: https://www.thermofisher.com/ch/en/home/references/ambion-tech-support/nuclease-enzymes/general-articles/dnase-i-demystified.html
It seem that both Ca and Mg are important for the activity.
thanks for this suggestion! I did not think to DNAses when asking, rather to nuclear membrane disruption, but you're right I need to take this into consideration.
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