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!

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