Problems with detecting LC3 in mammalian cell culture

[jl5zc]

I've been working on this Western blot for LC3 the past 5 weeks, nothing has come through successfully. I am using MBL Anti-LC3 (PD014) and wonder if anybody out there has used this ab and is having problems detecting endogenous LC3 in MEFs or HeLa.

If it helps, this is how I conducted the experiment:

- Starvation assay 1h incubation in EBSS medium.
- Wash cells in PBS, lyse with 1% Triton X and place on rotator for 30mins in cold room. Spin down for 10 mins, collect supernatant.
- Prepare samples with 2X reducing sample buffer for running gel. Boil samples for 5mins. Spin down.
- Run samples on a 13% and also a gradient (6-15%) SDS gel at 60V o/n.
- Transfer onto PVDF 0.45micron membrane on wet transfer at 100V for 1h.
- Block with 5% milk+TBST for 1h.
- Incubate with primary ab o/n 1:1000 dil in TBST+NaN3.
- Wash 3x5mins in TBST.
- Incubate with rabbit secondary ab 1:10,000 in 5% milk for 1h.
- Wash 3x5mins in TBST.
- Develop with Supersignal Femto/Pico.

I see no bands corresponding to LC3-I (18kDa) or LC3-II (16kDa). Ponceau stain shows no protein bands below 75kDa marker.

Jianyi

[hafid]

I am using the anti-LC3 from another compagny.
In general you have to check your input, verify your protein concentration, and take care to lyse with low salt lysis buffer (tris Hcl pH 7.4, Nacl, NP-40, EDTA , PMSF and proteases inhibitor cocktail from sigma.).
It'a not easy to detect both form of LC3 but most of compagnies recommand to use 1/500 dilution.
In our lab , I block with 5% milk, incubate primary in 2.5% milk, wash 3X 10 min in TBST, secondary in 2.5% milk and wash 5X 10 min ...
try to expose your film at 2 min and 10 min, because you have differents bands that are non specific that can be detected strongly.

[Cancerinform]

You should not spin down the insoluble fraction after lysing the cells. Also I compared several glioblastomas next to each other. Some cells have a lot of LC3 and other one can hardly detect any.