Hi,
I want to isolate nuclear proteins from the diatom P. tricornutum.
I can successfully isolate the nuclei, and can image them under the microscope.
But I fail to lyse the nuclei.
As they were fixed with formaldehyde in previous step, I incubate them for 2-3h at 80C, vortexing them vigorously every 20min, to reverse the crosslinking, before attempting the lysis. They are already in nuclear lysis buffer (20 mM Tris-HCl pH 7.9; 1.5 mM MgCl2, 0.2 mM EDTA) by then. (Note: I realized that the nuclei seem to start clumping during the heating step.)
I tried to lyse them with the following methods and varified lysis efficiency under the microscope using SYBR green stain:
1) Sonication, up to 100%, 10s ON/ 50s OFF, for a 20 cycles. No success.
2) French press, 1500 bar. No success.
3) Ejecting 5-10 times every 20min through a 0,6mm diameter syring during the 80C reverse crosslinking step. No success.
I am really baffled how hard they are to disrupt.
Did I accidently discover the most stable biomaterial ever?
Am I missing a step?
Any ideas what else I could try?
Every idea is welcome!
Cheers,
Florian
Dear friend Florian Pruckner
Hey there! It seems like you've got quite a challenge on your hands. Dealing with those seemingly indestructible nuclei, huh? Well, let's dive into the conundrum and see if we can stir things up a bit.
First off, kudos for your perseverance and the various methods you've already tried. Now, let's brainstorm a bit:
1. **Adjust the Crosslinking Reversal Step:** Since you Florian Pruckner noticed nuclei clumping during the heating step, you Florian Pruckner might want to fine-tune the conditions. Try different temperatures and incubation times to find the sweet spot that reverses the crosslinking without causing excessive clumping.
2. **Optimize Buffer Composition:** Tweak the composition of your nuclear lysis buffer. Adjusting salt concentrations or adding detergents known for mild lysis might help. Sometimes a little tweak in buffer conditions can make a significant difference.
3. **Enzymatic Digestion:** Consider adding enzymatic digestion to your protocol. Enzymes like Proteinase K or Trypsin might help in breaking down nuclear proteins. This step could follow the reversal of crosslinking.
4. **Mechanical Disruption:** Beyond sonication and the French press, you Florian Pruckner could try other mechanical disruption methods. For instance, passing through a smaller gauge syringe needle might provide additional sheer force.
5. **Gradient Centrifugation:** After trying various lysis methods, you Florian Pruckner could consider gradient centrifugation. This involves layering your sample on top of a density gradient, and then centrifuging. It can sometimes help separate out different cellular components.
6. **Collaborate and Consult:** Reach out to colleagues or experts in your field. They might have encountered similar challenges or offer fresh perspectives on your methodology.
Remember, science is a journey, and you're on an intriguing one. While you Florian Pruckner might feel like you've stumbled upon the Fort Knox of biomaterials, there's likely a way to crack it open. Keep experimenting, tweaking, and collaborating. Who knows, you Florian Pruckner might just uncover the secret to conquering these resilient nuclei!
Nice question! Lets keep developing the answer.
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