I am using these cell lines: HCT116 and HT29 (colorectal cancer cell lines) and BJ (skin fibroblasts) and IEC-6 (small intestine cells from rats).
We see LC3-II with different buffer,s either RIPA (with 0.1% SDS) or NP-40 (1%)-based buffer.
Hi
you can follow this protocol any human cells or mouse cells or tissues in western blot exp
Cell lysate preparation
Rippa buffer
1. Take 10 Rippa buffer in 15 tube and add one tablet of Protease inhibitor cocktail
2. Mix or vortex until tablet dissolves, aliquates in 1.5 ml microfuge tube and store -80°C
Suspension cells.
1. Harvest ells in 15 ml conical tube and centrifuge for 10 min at 1200 rpm
2. Remove the medium and add 1 ml of PBS transfer to 1.5 ml microfuge tube.
3 .Centrifuge 3min at 5000Rpm and remove the supernatant carefully.
4 .Remove the supernatant carefully without disrupting pellet.
5. Add rippa buffer 100 to 200ul and mix incubate ice 30 min, every 10 min vortex.
6. Sonicate 15 second with ice and Centrifuge at 12,000 rpm for 15 min at 4°C.**
7. Add 4 x buffers in the tube and heat the sample 100 °C for 3min.
8. Vortex and centrifuge 3 min 5000 rpm ready to load sample or store -80°C
For Adherent cells.
1. Remove the medium from plate and wash with 1 ml of 1X PBS.
2. Add 1 ml of PBS and scrape cells with cell scraper.
3. Transfer in to 1.5 ml microfuge tube and centrifuge 3min at 5000Rpm.
4. Remove the supernatant carefully without disrupting pellet.
5. Add rippa buffer 100 to 200ul and mix incubate ice 30 min, every 10 min vortex.
6. Sonicate 15 second with ice and Centrifuge at 12,000 rpm for 15 min at 4°C.**
7. Add 4 x buffers in the tube and heat the sample 100 °C for 3min.
8. Vortex and centrifuge 3 min 5000 rpm ready to load sample or store -80°C
** Take small volume of lysate and estimate protein amount
Reagents
BCA protein kit or thermo fisher kits
Rippa Buffer Cat#Prod89900 from Thermo fisher
Protease inhibitor cocktail tablets from Roche Diagonastic
I use a simple SDS-buffer: 50mM Tris pH7.5, 150mM NaCl, 1%SDS, 5mM EDTA.
Before adding to cells, I add 1ul/ml Benzonase to degrade DNA. Works well at 4C. Scrape adherent cells in this, add Sample buffer and bio. No need to centrifuge. Works very well for extraction of TCL. Run lysates on a gradient or 12 to 15% gel and you will get good LC3I and LC3II bands.
Hi,
I found out that I have a lot of LC3-II with my treatment after the protein extraction in the insoluble pellet (not that much in the control), so I was planning to change my protein extraction method so that I could have the whole LC3-II population as soluble form. (So far I have used RIPA including 0.1% SDS).
So, for extraction of LC3-II, I found several protocols that mainly involved heating in 1% SDS, sonication and centrifugation. Anyway, I would have one question. 1% SDS precipitates in the cold. Should I perform the final centrifugation at +4C or at RT? Centrifugation will pellet the SDS precipitate, but any clue whether it contains also proteins? (I can of course try it out...)
Thanks in advance!
^ Unfortunately, as I explained, in my case RIPA is not strong enough to solubilize total cellular LC3-II. In the soluble fraction, the difference in LC3-II between treatment and vehicle control is rather small or non-existing. However, in the insoluble pellet, there is much more LC3-II in the treatment samples versus vehicle.
Sonja, were you able to find a better way to solubilize LC3-II? If you were, could you please share your tricks:) Thanks!
Hi,Sonjia,have you ever compared control sample with chloroquine only?With RIPA(including 0.1%SDS and 1%Trition)?
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