LC3B Recombinant Rabbit Monoclonal Antibody [JJ090-6] - BSA and Azide free
Catalog# HA750319
LC3B Recombinant Rabbit Monoclonal Antibody [JJ090-6] - BSA and Azide free
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WB
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IF-Cell
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IHC-P
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IF-Tissue
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IP
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Human
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Mouse
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Rat
Overview
Product Name
LC3B Recombinant Rabbit Monoclonal Antibody [JJ090-6] - BSA and Azide free
Antibody Type
Recombinant Rabbit monoclonal Antibody
Immunogen
Synthetic peptide within human LC3 B aa 1-20.
Species Reactivity
Human, Mouse, Rat
Validated Applications
WB, IF-Cell, IHC-P, IF-Tissue, IP
Molecular Weight
Predicted band size: 14/16 kDa
Positive Control
HeLa cells treated with 50μM Chloroquine for 24 hours, HeLa cell lysate, HeLa treated with 50μM Chloroquine for 18 hours cell lysate, C2C12 cell lysate, C2C12 treated with 50μM Chloroquine for 18 hours cell lysate, C6 cell lysate, C6 treated with 50μM Chloroquine for 18 hours cell lysate, mouse brain tissue lysate, rat brain tissue lysate, HCT 116 cell lysate, HCT 116 treated with 50μM Chloroquine for 18 hours cell lysate, U-87 MG cell lysate, C2C12 cells treated with 50μM Chloroquine for 24 hours, C6 cells treated with 50μM Chloroquine for 24 hours, mouse brain tissue, mouse hippocampus tissue, rat brain tissue, rat hippocampus tissue.
Conjugation
unconjugated
Clone Number
JJ090-6
Product Features
Form
Liquid
Concentration
1ug/ul
Storage Instructions
Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage Buffer
PBS (pH7.4).
Isotype
IgG
Purification Method
Protein A affinity purified.
Application Dilution
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WB
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1:2,000-1:5,000
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IF-Cell
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1:100-1:200
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IHC-P
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1:1,000-1:5,000
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IF-Tissue
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1:500-1:1,000
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IP
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Use at an assay dependent concentration.
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mIHC
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1:100
Target
Function
Microtubule-associated proteins (MAPs) regulate microtubule stability and play critical roles in neuronal development and in maintaining the balance between neuronal plasticity and rigidity. MAP-light chain 3 beta (MAP-LC3β) and MAP-light chain 3 alpha (MAP-LC3α) are subunits of both MAP1A and MAP1B. MAP-LC3β, a homolog of Apg8p, is essential for autophagy and associated to the autophagosome membranes after processing. Two forms of LC3β, the cytosolic LC3-I and the membrane-bound LC3-II, are produced post-translationally. LC3-I is formed by the removal of the C-terminal 22 amino acids from newly synthesized LC3β, followed by the conversion of a fraction of LC3-I into LC3-II. LC3 enhances fibronectin mRNA translation in ductus arteriosus cells through association with 60S ribosomes and binding to an AU-rich element in the 3′ untranslated region of fibronectin mRNA. This facilitates sorting of fibronectin mRNA onto rough endoplasmic reticulum and translation. MAP LC3β may also be involved in formation of autophagosomal vacuoles. It is expressed primarily in heart, testis, brain and skeletal muscle.
Background References
1. Gai Z et al. Farnesoid X Receptor Protects against Kidney Injury in Uninephrectomized Obese Mice. J Biol Chem 291:2397-411 (2016).
2. Xu TX et al. Hypoxia responsive miR-210 promotes cell survival and autophagy of endometriotic cells in hypoxia. Eur Rev Med Pharmacol Sci 20:399-406 (2016).
Sequence Similarity
Belongs to the ATG8 family.
Tissue Specificity
Most abundant in heart, brain, skeletal muscle and testis. Little expression observed in liver.
Post-translational Modification
The precursor molecule is cleaved by ATG4B to form the cytosolic form, LC3-I. This is activated by APG7L/ATG7, transferred to ATG3 and conjugated to phospholipid to form the membrane-bound form, LC3-II.; The Legionella effector RavZ is a deconjugating enzyme that produces an ATG8 product that would be resistant to reconjugation by the host machinery due to the cleavage of the reactive C-terminal glycine.; Phosphorylation at Thr-12 by PKA inhibits conjugation to phosphatidylethanolamine (PE) (By similarity). Interaction with MAPK15 reduces the inhibitory phosphorylation and increases autophagy activity.
Subcellular Location
Cytoplasm, Cytoplasmic vesicle, Cytoskeleton, Membrane, Microtubule, Mitochondrion.
Synonyms
ATG8F antibody
Autophagy-related protein LC3 B antibody
Autophagy-related ubiquitin-like modifier LC3 B antibody
LC3B antibody
LC3II antibody
MAP1 light chain 3 like protein 2 antibody
MAP1 light chain 3-like protein 2 antibody
MAP1A/1BLC3 antibody
MAP1A/MAP1B LC3 B antibody
MAP1A/MAP1B light chain 3 B antibody
ExpandImages
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☑ Cell treatment (CT)
Western blot analysis of LC3B on different lysates with Rabbit anti-LC3B antibody (HA750319) at 1/2,000 dilution and competitor's antibody at 1/1,000 dilution.
Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: HeLa treated with 50μM Chloroquine for 18 hours cell lysate (20 µg/Lane)
Lane 3: C2C12 cell lysate (20 µg/Lane)
Lane 4: C2C12 treated with 50μM Chloroquine for 18 hours cell lysate (20 µg/Lane)
Lane 5: C6 cell lysate (20 µg/Lane)
Lane 6: C6 treated with 50μM Chloroquine for 18 hours cell lysate (20 µg/Lane)
Lane 7: mouse brain tissue lysate (20 µg/Lane)
Lane 8: rat brain tissue lysate (20 µg/Lane)
Predicted band size: 14/16 kDa
Observed band size: 14/16 kDa
Exposure time: 3 minutes;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA750319) at 1/2,000 dilution and competitor's antibody at 1/1,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature. -
☑ Knockdown (KD)
Western blot analysis of LC3B on different lysates with Rabbit anti-LC3B antibody (HA750319) at 1/2,000 dilution.
Lane 1: HeLa-si NT cell lysate (10 µg/Lane)
Lane 2: HeLa-si LC3B cell lysate (10 µg/Lane)
Predicted band size: 14/16 kDa
Observed band size: 14/16 kDa
Exposure time: 1 minute; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA750319) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature. -
☑ Cell treatment (CT)
Western blot analysis of LC3B on different lysates with Rabbit anti-LC3B antibody (HA750319) at 1/2,000 dilution.
Lane 1: HCT 116 cell lysate
Lane 2: HCT 116 treated with 50μM Chloroquine for 18 hours cell lysate
Lane 3: U-87 MG cell lysate
Lane 4: C6 cell lysate
Lane 5: Mouse brain tissue lysate
Lane 6: Rat brain tissue lysate
Lysates/proteins at 20 µg/Lane.
Predicted band size: 14/16 kDa
Observed band size: 14/16 kDa
Exposure time: 26 seconds; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA750319) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature. -
☑ Cell treatment (CT)
Immunocytochemistry analysis of C2C12 cells treated with or without 50μM Chloroquine for 24 hours labeling LC3B with Rabbit anti-LC3B antibody (HA750319) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-LC3B antibody (HA750319) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution. -
☑ Cell treatment (CT)
Immunocytochemistry analysis of HeLa cells treated with or without 50μM Chloroquine for 24 hours labeling LC3B with Rabbit anti-LC3B antibody (HA750319) at 1/200 dilution.
Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-LC3B antibody (HA750319) at 1/200 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution. -
☑ Cell treatment (CT)
Immunocytochemistry analysis of C6 cells treated with or without 50μM Chloroquine for 24 hours labeling LC3B with Rabbit anti-LC3B antibody (HA750319) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-LC3B antibody (HA750319) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution. -
Immunohistochemical analysis of paraffin-embedded mouse brain tissue with Rabbit anti-LC3B antibody (HA750319) at 1/5,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA750319) at 1/5,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
Immunohistochemical analysis of paraffin-embedded mouse hippocampus tissue with Rabbit anti-LC3B antibody (HA750319) at 1/5,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA750319) at 1/5,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-LC3B antibody (HA750319) at 1/5,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA750319) at 1/5,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
Immunohistochemical analysis of paraffin-embedded rat hippocampus tissue with Rabbit anti-LC3B antibody (HA750319) at 1/5,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA750319) at 1/5,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
Fluorescence multiplex immunohistochemical analysis of human gastric cancer (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-LC3B (HA750319, Green), anti-CD31 (M1511-8, Red) and anti-CA-IX (ET1701-51, Yellow) on human gastric cancer. HRP Conjugated UltraPolymer Goat Polyclonal Antibody HA1119/HA1120 was used as a secondary antibody. The immunostaining was performed with the Sequential Immuno-staining Kit (IRISKit™MH900205). The section was incubated in three rounds of staining: in the order of ET1701-65 (1/100 dilution), M1511-8 (1/2,000 dilution) and ET1701-51 (1/100 dilution) for 20 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 30 mins at 95℃. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Olympus VS200 Slide Scanner.
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