LC3B Recombinant Rabbit Monoclonal Antibody [JJ090-6]
cat.: ET1701-65
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human, Mouse, Rat
Applications: WB, IF-Cell, IHC-P, IF-Tissue, IP
Clonality: Monoclonal
Clone number: JJ090-6
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 1ug/ul
Purification: Protein A affinity purified.
Molecular weight: Predicted band size: 14/16 kDa
Isotype: IgG
Immunogen: Synthetic peptide within human LC3 B aa 1-20 and aa 80-120.
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.
Subcellular location: Cytoplasm, Cytoplasmic vesicle, Cytoskeleton, Membrane, Microtubule, Mitochondrion.
Recommended Dilutions:
  WB
  IF-Cell
  IHC-P
  IF-Tissue
  IP

1:1,000-1:2,000
1:100-1:200
1:1,000-1:5,000
1:200-1:1,000
Use at an assay dependent concentration.
Uniprot #: SwissProt: Q9GZQ8 Human | Q9CQV6 Mouse | Q62625 Rat
Alternative names: ATG8F Autophagy-related protein LC3 B Autophagy-related ubiquitin-like modifier LC3 B LC3B LC3II MAP1 light chain 3 like protein 2 MAP1 light chain 3-like protein 2 MAP1A/1BLC3 MAP1A/MAP1B LC3 B MAP1A/MAP1B light chain 3 B MAP1ALC3 MAP1LC3B a Map1lc3b Microtubule associated protein 1 light chain 3 beta Microtubule-associated protein 1 light chain 3 beta Microtubule-associated proteins 1A/1B light chain 3B MLP3B_HUMAN
Images
ET1701-65_1.png Fig1: Immunocytochemistry analysis of HeLa cells treated with or without 50μM Chloroquine for 24 hours labeling LC3B with Rabbit anti-LC3B antibody (ET1701-65) 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 (ET1701-65) 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.
ET1701-65_2.jpg Fig2: Western blot analysis of LC3B on different lysates with Rabbit anti-LC3B antibody (ET1701-65) at 1/2,000 dilution and competitor's antibody at 1/1,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HeLa treated with 50μM Chloroquine for 18 hours cell lysate
Lane 3: C2C12 cell lysate
Lane 4: C2C12 treated with 50μM Chloroquine for 18 hours cell lysate
Lane 5: C6 cell lysate
Lane 6: C6 treated with 50μM Chloroquine for 18 hours cell lysate
Lane 7: mouse brain tissue lysate
Lane 8: rat brain tissue lysate
Lysates/proteins at 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 (ET1701-65) 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.
ET1701-65_3.jpg Fig3: Western blot analysis of LC3B on different lysates with Rabbit anti-LC3B antibody (ET1701-65) at 1/1,000 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;

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 (ET1701-65) at 1/1,000 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.
ET1701-65_4.jpg Fig4: Western blot analysis of LC3B on different lysates with Rabbit anti-LC3B antibody (ET1701-65) at 1/2,000 dilution.

Lane 1: HeLa-si NT cell lysate
Lane 2: HeLa-si LC3B cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 14/16 kDa
Observed band size: 14/16 kDa

Exposure time: 1 minute;

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 (ET1701-65) 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.
ET1701-65_5.jpg Fig5: Immunocytochemistry analysis of C2C12 cells treated with or without 50μM Chloroquine for 24 hours labeling LC3B with Rabbit anti-LC3B antibody (ET1701-65) 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 (ET1701-65) 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.
ET1701-65_6.jpg Fig6: Immunocytochemistry analysis of C6 cells treated with or without 50μM Chloroquine for 24 hours labeling LC3B with Rabbit anti-LC3B antibody (ET1701-65) 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 (ET1701-65) 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.
ET1701-65_7.jpg Fig7: Immunohistochemical analysis of paraffin-embedded mouse brain tissue with Rabbit anti-LC3B antibody (ET1701-65) 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 (ET1701-65) 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.
ET1701-65_8.jpg Fig8: Immunohistochemical analysis of paraffin-embedded mouse hippocampus tissue with Rabbit anti-LC3B antibody (ET1701-65) 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 (ET1701-65) 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.
ET1701-65_9.jpg Fig9: Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-LC3B antibody (ET1701-65) 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 (ET1701-65) 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.
ET1701-65_10.jpg Fig10: Immunohistochemical analysis of paraffin-embedded rat hippocampus tissue with Rabbit anti-LC3B antibody (ET1701-65) 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 (ET1701-65) 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.
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