LC3B (D11) XP® Rabbit mAb (Biotinylated)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Lyse cells in RIPA buffer (2 mM pH 8.0 ETDA, 50 mMTris pH 7.4, 150 mMNaCl, 1% NP-40, 1 mM PMSF, 1% sodium deoxycholate) containing protease inhibitor cocktail	- Dilute Ab at 1:1000 and incubate overnight at 4°C with primary antibody

Upstream tips
- Lyse cells in RIPA buffer (2 mM pH 8.0 ETDA, 50 mMTris pH 7.4, 150 mMNaCl, 1% NP-40, 1 mM PMSF, 1% sodium deoxycholate) containing protease inhibitor cocktail
Protocol tips
- Dilute Ab at 1:1000 and incubate overnight at 4°C with primary antibody

Publication protocol

Following treatment protocols, cells were washed twice with ice-cold PBS (2.7 mMKCl, 2 mM KH2PO4, 137 mMNaCl, 10 mM Na2HPO4, pH 7.4), then lysed with RIPA buffer (2 mM pH 8.0 ETDA, 50 mMTris pH 7.4, 150 mMNaCl, 1% NP-40, 1 mM PMSF, 1% sodium deoxycholate) containing protease inhibitor cocktail (Roche, 04693116001). The concentration of protein extracts was assayed with Bio-Rad Protein Assay Kit (Bio-Rad, 500-0006). Cell lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to hydrophobic polyvinylidene fluoride transfer membrane (PVDF) (PALL, 29301-808). Membranes were blocked in TBS containing 5% non-fat milk and 0.2% Tween 20 for 1 h and probed overnight at 4°C with primary antibodies. Antibodies against HK2 (2867), PARP (9542), LC3B (3868), cleaved caspase-7 (9491), cleaved caspase-3 (9661), p-AKTSer473 (9271) and p-AMPKαThr172 (2535) were obtained from Cell Signaling Technology. Antibody against p-p70S6kThr389 (MABS82) was purchased from Millipore. Antibody against LCN2 (AF1757) was supplied by R&D Systems and β-actin (A5441) was supplied by Sigma. Blots were washed in 0.2% TBS-Tween 20 and incubated with HRP-conjugated secondary antibodies for 1 h. The protein blots were visualized using enhanced chemiluminescence reagent obtained from Perkin Elmer (NEL105001EA).

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Autophagy assay cell type - Beas-2B using LC3B (D11) XP® Rabbit mAb (Biotinylated) from Cell Signaling Technology.

Manufacturer protocol

Download the product protocol from Cell Signaling Technology for LC3B (D11) XP® Rabbit mAb (Biotinylated) below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Autophagy assay cell type - Beas-2B using LC3B (D11) XP® Rabbit mAb (Biotinylated) from Cell Signaling Technology. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.