Anti lc3 antibody

Manufactured by Abcepta

Sourced in United States

The Anti-LC3 antibody is a protein used in research applications to detect and quantify the presence of the LC3 (Microtubule-Associated Protein 1 Light Chain 3) protein. LC3 is a widely used marker for monitoring autophagy, a cellular process involved in the degradation and recycling of damaged or unnecessary cellular components.

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Rabbit anti-LC3 polyclonal antibody Anti-LC3

3 protocols using anti lc3 antibody

Antibody Sources for Protein Detection

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Dulbecco’s modified Eagle’s medium (DMEM), penicillin, and streptomycin were purchased from Cibco BRL (Paisley, Scotland, U.K.). Dimethyl sulphoxide (DMSO) and EDTA were purchased from Sigma Chemical (Poole, Dorset, U.K.). Potassium dichromate (K₂Cr₂O₇) and NAC were obtained from Merck Chemical Co. (Darmstadt, Germany). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from Sigma-Aldrich, Inc. (St. Louis, MO, U.S.A.). The antibodies for detecting anti-poly-(ADP-ribose) polymerase (PARP) antibody were obtained from Millipore (Billerica, MA, U.S.A.); anti-caspase-3 and anti-cleaved-caspase-3 antibodies were obtained from Epitomics (Burlingame, CA, U.S.A.); anti-LC3 antibody was obtained from Abgent (San Diego, CA, U.S.A.). Akt, phospho-Akt, p65, phospho-p65, IκB-α, ERK1/2, phospho-ERK1/2, p-38, phospho-p38, JNK and phospho-JNK were purchased from Cell Signaling (Beverly, MA, U.S.A.); GAPDH was obtained from Abcam Inc.(Cambridge, MA, U.S.A.); phospho- IkB-α was purchased from Upstate Biotechnology (Lake Placid, NY, U.S.A.). Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, U.S.A.). TNF-α and IL-1α antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).

Lee Y.H., Su S.B., Huang C.C., Sheu H.M., Tsai J.C., Lin C.H., Wang Y.J, & Wang B.J. (2014). N-Acetylcysteine Attenuates Hexavalent Chromium-Induced Hypersensitivity through Inhibition of Cell Death, ROS-Related Signaling and Cytokine Expression. PLoS ONE, 9(9), e108317.

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Immunofluorescence Assay for LC3 Detection

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AMϕ were cytospun onto a microscope slide and fixed with 4% paraformaldehyde for 20 min. After washing with PBS, the cells were permeabilized with 0.25% Triton X-100 in PBS for 10 min at room temperature, followed by blocking with 1% bovine serum albumin (BSA) in PBST (PBS with 0.2% Tween-20) for 2 h at room temperature to reduce nonspecific staining. The cells were then incubated with anti-LC3 antibody (1:500; Abgent, San Diego, CA) at 4°C overnight. After washing twice with PBS, the cells were incubated with Alexa Fluor 555-conjugated anti-rabbit IgG (1:500; Cell Signaling Technology, Beverly, MA) for 1 h at room temperature. Hoechst 33258 (1:200; Sigma-Aldrich, St. Louis, MO) was used to stain nuclei. The cells were then washed with PBS for 3 times, followed by confocal microscopy.

Wen Z., Fan L., Li Y., Zou Z., Scott M.J., Xiao G., Li S., Billiar T.R., Wilson M.A., Shi X, & Fan J. (2014). Neutrophils Counteract Autophagy-Mediated Anti-Inflammatory Mechanisms in Alveolar Macrophage: Role in Post-Hemorrhagic Shock Acute Lung Inflammation. Journal of immunology (Baltimore, Md. : 1950), 193(9), 4623-4633.

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Quantifying Autophagy Markers in ACC Cells

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The radioimmunoprecipitation assay (Sigma-Aldrich) lysate and phenylmethylsulfonyl fluoride (Sigma-Aldrich) were mixed to extract the total proteins from the SIL-treated ACC-M cells and the lung metastases of ACC nude mice. The bicinchoninic acid method was used to detect the total proteins. The proteins were then mixed with 2× sodium dodecyl sulfate loading buffer and heated until denaturation. Fifty micrograms of proteins was loaded into each lane for a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Afterward, the proteins were transferred onto one polyvinylidene difluoride membrane, followed by closure with 3% skimmed milk at room temperature overnight; the primary antibodies (anti-β-actin antibody (Abgent, San Diego, CA, USA): diluted with Tris-buffered saline and tween 20 (TBST), 1:2,000; anti-LC3 antibody (Abgent): diluted with TBST, 1:500) were then added and incubated at 4°C overnight. After washing with TBST three times (slightly pendulated for 10 minutes each time), an horse radish peroxidase-conjugated goat antimouse IgG (Abgent) (dilution with TBST, 1:2,000) was added and incubated at room temperature for 1 hour. After subsequently washing with TBST three times for 10 minutes, the ECL kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used for coloration and the films were then developed in darkness.

Jiang C., Jin S., Jiang Z, & Wang J. (2016). Inhibitory effects of silibinin on proliferation and lung metastasis of human high metastasis cell line of salivary gland adenoid cystic carcinoma via autophagy induction. OncoTargets and therapy, 9, 6609-6618.

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