Protocol: Inhibition of Autophagy and LC3 Antibody (NBP1-19167) Western Blot
Chloroquine diphosphate (CQ) (10 mM) in dH2O
Sample buffer, 2X Laemmli buffer: 4% SDS, 5% 2-mercaptoethanol (BME), 20% glycerol, 0.004% bromophenol blue, 0.125 M Tris HCl, pH 6.8
RIPA buffer: 150 mM NaCl, 1% NP-40 or Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0, 20 mM Tris-HCl, pH 7.5
1X Running Buffer: 25 mM Tris-base, 192 mM glycine, 0.1% SDS. Adjust to pH 8.3
1X Transfer buffer (wet): 25 mM Tris-base, 192 mM glycine, 20% methanol, Adjust to pH 8.3
TBST, TBS and 0.1% Tween
Blocking solution: TBST, 5% non-fat dry milk
rabbit anti-LC3 primary antibody (NBP1-19167) in blocking buffer (~2 ug/mL)
Tip: For more information on Western Blotting, see our Western Blot handbook.
1. Grow cells (e.g. HeLa or Neuro2A) in vitro to semi-confluency (70-75%).
2. Add CQ to culture dishes to a final concentration of 50 uM and incubate overnight (16 hours). Remember to include an untreated sample as a negative control.
Note: Validated autophagy inducers should be included as positive controls.
3. Rinse cells with ice-cold 1X PBS and lyse cells with sample buffer.
Note: LC3-I and LC3-II are sensitive to degradation, although LC3-I is more labile. These proteins are sensitive to freeze-thaw cycles and SDS sample buffers. Fresh samples should be analyzed quickly to prevent protein degradation.
4. Sonicate and incubate cells for 5 minutes at 95oC.
Tip: Cells are lysed directly in sample buffer or may be lysed in RIPA buffer.
5. Load samples of Chloroquine-treated and -untreated cell lysates 40 ug/lane on a 4-20% polyacrylamide gradient gel (SDS-PAGE).
Tip: For detection of LC3 it is particularly important to monitor the progress of the gel as this protein is relatively small (~14kDa).
Tip: Alternatively, for non-gradient gels, use a 20% polyacrylamide gel.
6. Transfer proteins to a 0.2 um PVDF membrane for 30 minutes at 100V.
7. After transfer, rinse the membrane with dH2O and stain with Ponceau S for 1-2 minutes to confirm efficiency of protein transfer.
8. Rinse the membrane in dH2O to remove excess stain and mark the loaded lanes and molecular weight markers using a pencil.
9. Block the membrane using blocking buffer solution (5% non-fat dry milk in TBST) for 1 hour at room temperature.
10.Rinse the membrane with TBST for 5 minutes.
11.Dilute the rabbit anti-LC3 primary antibody (NBP1-19167) (~2 ug/mL) in blocking buffer and incubate the membrane for 1 hour at room temperature.
12.Rinse the membrane with dH2O.
13.Rinse the membrane with TBST, 3 times for 10 minutes each.
14.Incubate the membrane with diluted secondary antibody, according with product's specifications, (e.g. anti-rabbit-IgG HRP-conjugated) in blocking buffer for 1 hour at room temperature.
Note: Tween-20 may be added to the blocking or antibody dilution buffer at a final concentration of 0.05-0.2%, provided it does not interfere with antibody-antigen binding.
15.Rinse the membrane with TBST, 3 times for 10 minutes each.
16.Apply the detection reagent of choice (e.g. BioFX Super Plus ECL) in accordance with the manufacturer's instructions.
17.Image the blot.
Tip: LC3-I and it's lipidated form LC3-II have different electrophoretic mobility properties, with the lipidated form moving faster in an SDS-PAGE gel, albeit its larger molecular weight. LC3-II runs at 14-16 kDa while LC3-I runs at 16-18kDa.
Note: This assay measures the difference in the LC3-II signal in the presence and absence of inhibitors (e.g., lysosomotropic agents). When autophagic flux is present or induced in a system an increase in the LC3-II signal should be observed with the inhibitor.