I've recently followed two different protocols which are used to extract Nuclei for immunolocalisation. But both quality and quantity are very poor having with much cell debris. Does anyone have any effective protocol for Nuclei isolation from Arabidopsis thaliana leaves?
Thanks a lot for any suggestions.
I have used C-TAB method for genomic DNA isolation from Arabidopsis. It worked great. I can share the protocol with you.
Thanks Shailja, it would be great if you can share it. Have you also checked the quality and concentration of nuclei using your protocol? or it's just only for genomic DNA ?
Here is our protocol, it works great, you need more than 1g of tissue however:
Cellular Distribution of snc1-GFP
Three-week-old plants (0.5 g) were harvested and ground to a fine
powder in liquid nitrogen and mixed with 2 volumes of lysis buffer (20 mM
Tris-HCl, pH 7.4, 25% glycerol, 20 mM KCl, 2 mM EDTA, 2.5 mM MgCl2,
250 mM sucrose, and 1 mM PMSF). The homogenate was filtered through
a 95- and 37-mm nylon netting successively. The flow-through was spun
at 1500g for 10 min, and the supernatant consisting of the cytosolic
fraction was collected and mixed with 53 Laemmli loading buffer and
heated at 958C for 5 min. The pellet was washed four times with 5 mL of
nuclear resuspension buffer NRBT consisting of 20 mM Tris-HCl, pH 7.4,
25% glycerol, 2.5 mM MgCl2, and 0.2% Triton X-100. The final pellet was
mixed with 50 mL of 13 Laemmli buffer and heated at 958C for 5 min. Fifty
microliters of each fraction was loaded on an 8% SDS-PAGE gel for
protein separation. Antibodies used for immunoblot analyses were as
described: anti-Histone H3 (Feys et al., 2005), anti-GFP (Wirthmueller
et al., 2007), and anti-PEPC (Noe¨ l et al., 2007). Band intensities were
measured by Quantity One 4.6.1 software (Bio-Rad). The experiment was
repeated five times; a figure representative of all repetitions is shown.
Various exposure times on films were used to make sure that the
exposure used was in the linear range.
Please cite: Cheng Y.*, Germain H.*, Wiermer M.*, Bi D.*, Xu F., García A., Wirthmueller L., Després C., Parker J., Zhang Y., and Li X. 2009. Nuclear Pore Complex Component MOS7/Nup88 Is Required for Innate Immunity and Nuclear Accumulation of Defense Regulators in Arabidopsis. THE PLANT CELL, 21(8) 2503-2516.
You can firstly get the protoplast by using cellulase and pectinase to treat the leaf of Arabidopsis, and then it is easy to wash the cell debris. when you acquired the protoblast, used some nuclei extration buffers to isolation the purified nuclei.
Hello Surya,
Here is a detailed protocol.
· Grind the seedlings in liquid Nitrogen to a fine powder.
· Add the powder to 30 ml of Extraction Buffer 1 in a 50 ml falcon tube. After adding buffer ensure you mix well so all the tissue has dissolved into the buffer.
· Filter the solution through a double layer of Miracloth into a 50 ml falcon tube. Repeat until the solution is clear.
· Spin the filtered solution for 20 min at 5000 g in a centrifuge at 4°C.(Advice:This step pellets the nuclei and other organelles. Buffer 1 contains enough sucrose for the nuclei not to burst open.)
· Gently remove supernatant and resuspend the pellet in 1 ml of Extraction Buffer 2. Transfer the solution to 1.5 ml eppendorf tube.
· Centrifuge at 12,000 g for 10 min at 4 °C.(Advice:If the protocol is working well, this pellet sho uld have a white fleck (nuclei). If not, don’t worry - it can be hidden under the other tissue. Buffer 2 contains enough detergents to burst open the chloroplasts but hopef ully not the nuclei.)
· Remove supernatant and resuspend pellet in 300 ul of Extraction Buffer 3.(Advice:The pellet can be diffic ult to resuspend at this point. Don’t worry some particle may not resuspend no matter how much you try!)
· In a clean eppendorf, add 900 ul of Extraction Buffer 3. Take the 300 ul solution(resuspended pellet) from step 19 and caref ully layer it on top of the clean 900 ul of Extraction Buffer 3.(Advice:It is best to slowly drop the resuspended pellet down the side of the eppendorf. This step just sets up a rough sucrose gradient so when you spin down the nuclei sho uld pellet and the chloroplasts end up in the upper layer)
· Spin for 1 hour at 16,000 g at 4 °C.
· Remove the supernatant and resuspend the chromatin pellet in 300 ul of Nuclei Lysis Buffer.
Extraction Buffer 1: 0.4 M sucrose, 10 mM Tris-HCl pH 8, 10 mM MgCl2 , 5 mM BME, 2 complete tablets / 100 ml 2 tablets
Extraction Buffer 2:0.25 M sucrose 1.25 ml 2 M, 10 mM Tris-HCl pH 8 , 10 mM MgCl2 , 1% Triton X-100 , 5 mM BME, 1 complete mini
Extraction Buffer 3: 1.7 M sucrose, 10 mM Tris-HCl pH 8, 2 mM MgCl2, 0.15% Triton X-100, 5 mM BME, 1 complete mini
Nuclei Lysis Buffer (Prepare fresh): 50 mM Tris-HCl pH8, 10 mM EDTA,1% SDS,1 complete mini 1 ml
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