I am trying nucleolar isolation from hela cells.
Do you have any specific questions? Do you have kits available to you? Otherwise, a good, generalized protocol can be found here:
http://www.lifetechnologies.com/us/en/home/references/protocols/cell-and-tissue-analysis/elisa-protocol/elisa-sample-preparation-protocols/nuclear-extraction-method-.html
I've had OK success with the protocol and it circumvents the need to buy kits, though those admittedly may give better results.
From the page (buffer recipes at that page):
1. Collect cells (5 x 106) in PBS by centrifugation (non-adherent) or scraping from culture flasks (adherent).
2. Wash cells twice with cold PBS.
3. Remove and discard the supernatant and collect the cell pellet.
4. Transfer the cells into a prechilled microcentrifuge tube.
5. Gently resuspend cells in 500 μl 1x Hypotonic Buffer by pipetting up and down several times. Incubate on ice for 15 minutes.
6. Add 25 μl detergent (10% NP40) and vortex for 10 seconds at highest setting.
7. Centrifuge the homogenate for 10 minutes at 3,000 rpm at 4°C.
8. Transfer and save the supernatant. This supernatant contains the cytoplasmic fraction. The pellet is the nuclear fraction.
9. Resuspend nuclear pellet in 50 μl complete Cell Extraction Buffer for 30 minutes on ice with vortexing at 10 minute internals.
10. Centrifuge for 30 minutes at 14,000 x g at 4°C. Transfer supernatant (nuclear fraction) to a clean microcentrifuge tube.
11. Aliquot and store at –80°C. The nuclear extracts are ready for assay.
You may see Nuclei Isolation Kit : Nuclei EZ Prep available from Sigma-Aldrich for details.
I am working on Abaqus/Explicit(Quasistatic ) for the deformation of the auxetic structure model. Please explain how the plastic input value should be considered from the true stress-strain curve...
05 August 2024 454 3 View
In Zn-Air Battery, how to plot Potential vs current density(mA/cm-2) in EC-Lab softare/BioLogic instrument. Please suggest. plot of the ref....
04 July 2024 3,807 2 View
and will i add phytochemical along with FUDR?
28 June 2024 6,254 0 View
As many of us know, there is often confusion regarding the alignment of dates, months, and seasons in our agricultural practices. To address this, I have prepared a table that aims to provide a...
27 June 2024 7,749 1 View
In Zn-Air Battery experiment, how to plot Potential vs current density(mA/cm-2) from discharge voltage curves. Please suggest.
23 June 2024 860 2 View
A masonry structure modelled all the wall using macro element and performing non linear time history analysis in dynamic explicit . I need to determine the random cracks where a give stress...
01 June 2024 1,349 1 View
Hi, I want to calculate the plastic equivalent strain for Mohr-Coulomb. I found a formula from Abaqus documentation. I have attached the photo of PEEQ calculation. But I want to figure out the...
28 May 2024 9,081 1 View
If the Fuzzy logic controller is not giving the desired results than what to do ? Further more...How to know how many membership functions and which type should be chosen?
20 May 2024 628 0 View
My student is isolating phages against ESKAPE pathogens, but she is getting a minute plaque. Can you please suggest how to get better results?
07 May 2024 9,554 2 View
I have added references using Mendeley reference manager, but it ruined the manuscript; it doesn't take ACS or other journals references properly except elsevier, and multiple duplicates are...
22 April 2024 6,560 1 View
Hello, I am currently having problems with RNA extraction. I am using mouse liver (C57BL6J), and I have extracted RNA from mouse liver before. Before this experiment, my final RNA pellets were...
11 August 2024 7,082 3 View
I have been doing the m6A dot blot for a while with no improvement, I am extracting the RNA, and I can see the dots although the three biological replicas give a different reading on the memberan...
10 August 2024 8,539 5 View
I have carried out MFC experiments on three different volumes, 50, 500 and 1000 mL of wastewater. Results after MFC treatment shows that TDS and EC are more in larger volumes of water i.e. TDS and...
09 August 2024 9,621 0 View
Hello everyone! I observed in my culture (htert-RPE1 cells) an orange- red particle at the bottom of the dish. It is visible to the naked eye as a very very small red dot. Could it be a...
09 August 2024 2,824 3 View
This larva was captured using a plankton net in the Persian Gulf during the summer. I believe it may be a Facetotecta nauplius.
08 August 2024 3,746 4 View
May members post flyers about opportunities to present at a conferehttps://veraeducation.com/nce? If so, where to post for the Virginia Educational Research Association? https://veraeducation.com/
08 August 2024 4,585 1 View
I've been performing RNA extraction on cotton petiole tissue for a few months now using the method described in the following paper, a derivative of the typical hot borate method...
08 August 2024 9,882 2 View
I am currently working on LncRNA; to know the lncRNA-protein interactions I want to do RNA pull down assay, so I need to design primers with T7 promoter. I need assistance in this regard.
07 August 2024 6,622 1 View
I am working in fungal fermentation of soybean meal and there is bacterial growth in them at times. I am trying to quantify fungal cell counts and bacterial cells; but I haven't been able to do at...
07 August 2024 7,535 4 View
My name is Apurva Saoji. I am a Ph.D scholar in Computer engineering in India. I am looking for international expert in reviewing my PhD thesis, "Competitive Optimization Techniques to Minimize...
07 August 2024 4,600 2 View