Hi, I am having so so so much trouble getting LC3 on WB.
Here is what I have been doing:
Conditions:
HeLa cells, AA starvation, EBSS, Rapamycin, Torin 1. They are all with/without NH4Cl+Leupeptin or Pepstatin A.
Cell lysis:
I tried 3 different ways.
1) RIPA 25mM Tris pH 7.6, 100mM NaCl, 0.5% NP40, 0.5% Na Deoxycholate, 50mM NaF, Complete protease tablet, 30min on ice, no sonication/ or sonication with water bath sonicator 10sec x 4, 1min rest in between. 13krpm spin for 20min and only SUP used. (Pellet was used once and it gave some faint signal)
2) RIPA 25mM Tris pH 7.6, 150mM NaCl, 1% NP40, 1% Na Deoxycholate, 0.1% SDS, Complete protease tablet. 30 min on ice, sonicate for 15sec using immersion sonicator, spin 12krpm, 15min.
3) RIPA 25mM Tris pH 7.6, 150mM NaCl, 1% NP40, 1% Na Deoxycholate, 0.1% SDS, Complete protease tablet, PMSF, Benzonase. 30 min on ice, sonicate for 15sec using immersion sonicator, spin 12krpm, 15min.
SDS page:
12% gel, 30ug protein/lane and run was stopped 1.5cm before the end. For lysis 2) and 3), 80ug protein/lane was used.
Transfer:
Semi-dry transfer by BioRad, Filters soaked in 25mM Tris 192mM glycine 20% methanol for 20min. 4 layers of filter on each side used for transfer. Transfer at 25V 350mA 90 min. PVDF membrane used after 10min soaking in methanol, quick rince in water and transfer buffer. Ponceus shows nice transfer all the way from 10kDa to 100kDa.
Antibody used:
Novus biochemicals the famous NB100-2220 Lot CW at 1/500 O/N 4C.
Blocking 5%Skim milk 1% BSA in TBS pH7.6 1hour, LC3 antibody O/N, 3x 10min rince with TBS pH 7.6 Tween20 0.1%, Secondary is PA-HRP bioRad 1/1000 45min-1hour RT. Rince 3x10min, visualize with Perkin Elmer ECL Plus kit (1:1 mix). Blot is never let dry.
2,5,10, and even 30 min doesn't reveal much band. At first I was getting strong band at 50kDa (and weak band around 15kDa) so I cut the membrane below 37kDa. Even this didn't reveal much LC3 around 15kDa.
What am I doing wrong? How can it not work at all?
Previously I was getting some signal in mCherry-LC3 transiently transfected HeLa cells, but I need to detect the endogeneous LC3. Please help me!!
If anyone out there working at GLEN site of MUHC, Montreal QC and you can visualize LC3, please help! Thanks!
Your protocol to obtain proteins seems to be OK, how about the amount of protein loaded in SDS-PAGE? About 20-30ug is necessary. Generally, LC3-I (between 15 and 20kDa) is difficult to detect as compared with lipidated form of LC3-II (between 10 and 15kDa).
Please let me know the clone name of the antibody you use?
And the following literature by Profesor Mizushima would be helpful for you!
Article How to Interpret LC3 Immunoblotting
The things that you can change are
1. Use a 15 % gel
2. Run the gel slowly...50V for stacking, 100V for resolving with some cooling
3. run the gel till your 10hd ladder reaches the end of the gel.
4. I can see that you are using rapamycin and torin both of which are strong inducers of autophagy.
5. Try adding more protein upto 50ug
6 try adding chloroquine or bafilomycin a 4 hrs prior to ur stipulated end time of experiment to serve as positive controls.
I regularly use that antibody and I get super blots for LC3.
Hi, Thank you everyone for advice.
I was loading from 30, 50 and all the way up to 80ug/ lane in 12% gel. Unfortunately, I wasn't able to get any endogeneous LC3 signal from these cells although they have been induced autophagy and also supplied with protease inhibitors.
I am now running the transiently transfected HeLa cells (LC3) and they have been induced with various autophagy drugs and starvation conditions. At the same time, I have leupeptin+NH4Cl for 4 hours. I am also running the gel all the way to the end, and this time I have 50ug protein /lane. Hopefully this works! I will come back to update you all with the result. Thanks!
Hi, here I came back with my LC3 blot. This is the same antibody as above, just a different lot. I am getting around 52 and 42kDa bands in all lanes. First lane on the left 4 is the Bafilomycin treated. Four lanes on the left are the same with NH4Cl and Leupeptin as inhibitors. Since they are transfected with mCherry-LC3, I was expecting 46, 44kDa. As you can see, I am not getting any endogeneous bands! (50ug/lane, 15% gel). Blank lanes are not-transfected samples.
Since my 2 bands are off the expected, I can't even tell if there is LC3I->LC3II conversion at all!
Hi Margit, thanks for your answer. I am thinking maybe I can improve my blotting conditions. What are the conditions for your transfer? I am currently using 0.35mA 25V for 90 min for the semi dry. I used 12% and 15% gel to transfer at this condition. Thanks once again!
Hi Margit, I have tried many different tricks, and now it sort of works.
After lysing the cells, I measure protein concentration, and dilute them in Laemmli buffer right away. Then instead of boiling them at 95C for 5 min, I boil them at 70C for 10 min.
After running the gel and transferring (semi-dry, 0.4mA 25V 60min, with 10% MeOH), I use phosphotase inhibitors (sodium orthovanadate and sodium fluoride) in all subsequent solutions. Also, I changed my secondary antibody to something that is extremely efficient.
Now, I get nice bands. But a bit too many bands... The LC3 (15,17kda) shows up very nicely!
Thanks so much!
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