LC3A/B (D3U4C) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #13082

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Dilute primary antibody in 1% normal serum or BSA (in 1X PBS). Incubate coverslips with ~5 µ g/mL rabbit anti-LC3 primary antibody (NB100-2220) for 1 hour at room temperature (37°C is optional), or for 16 hours at 4°C.

Protocol tips
Dilute primary antibody in 1% normal serum or BSA (in 1X PBS). Incubate coverslips with ~5 µ g/mL rabbit anti-LC3 primary antibody (NB100-2220) for 1 hour at room temperature (37°C is optional), or for 16 hours at 4°C.

Publication protocol

Cells were lysed in 2 × SDS-sample buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 20% glycerol, 5% 2-mercaptoethanol, and 0.025% bromophenol blue). Protein concentrations were quantified with the Lowry assay using the RC DC protein assay kit (Bio Rad, 500-0120JA). Then, 20 μg of total protein were resolved by SDS-PAGE and transferred to PVDF membranes (Millipore, IPVH00010). Blotted membranes were incubated with 3% non-fat dry milk in PBS with 0.05% Tween-20 for 30 min to block nonspecific binding, and then the indicated antibodies were added for overnight incubation at 4°C. After 1 h incubation at room temperature with HRP-linked anti-rabbit IgG (GE Healthcare, NA9340V), protein bands were detected with ECL Prime (GE Healthcare, RPN 2232) and analyzed with LumiVision Pro (Aishin). The intensity of expression was analyzed with LumiVision Pro Analyzer 400 software (Aishin). Antibody to GADD34 (C-19, sc-825) was obtained from Santa Cruz. Anti-β-actin antibody was from Sigma (A2066). Anti-LC3 anbibody was from Novus (NB100-2220). Anti-phospho-Src family (Tyr 416, #2101), anti-Src (#2109), anti-phospho-p38 (Thr180/Tyr182, #9211), anti-p38 (#9212), anti-phospho-Erk1/2 (Thr202/Tyr204, #9101), anti-Erk1/2 (#9102), anti-phospho-Akt (Ser473, #9271), anti-caspase 3 (#9662), anti-phospho-mTOR (Ser2448, #5536), anti-mTOR (#2972), anti-phospho-p70 S6K (Thr389, #9205), anti-p70 S6K (#2708), anti-phospho-4E-BP1 (Thr37/46, #2855), anti-4E-BP1 (#9452), anti-phospho-TSC2 (Thr1462, #3611), anti-TSC2 (#4308), anti-NBR1 (#9891) and anti-GAPDH (#2118) were from Cell Signaling Technology.

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 - RAW 264.7 using LC3A/B (D3U4C) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #13082 from Cell Signaling Technology.

Manufacturer protocol

Download the product protocol from Cell Signaling Technology for LC3A/B (D3U4C) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #13082 below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Autophagy assay cell type - RAW 264.7 using LC3A/B (D3U4C) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #13082 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.