Validated using a knockout cell line
Recombinant
RabMAb

Anti-LC3B antibody [EPR18709] (ab192890)

Overview

  • Product name
    Anti-LC3B antibody [EPR18709]
    See all LC3B primary antibodies
  • Description
    Rabbit monoclonal [EPR18709] to LC3B
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IP, WB, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human LC3B aa 1-100. The exact sequence is proprietary.
    Database link: Q9GZQ8

  • Positive control
    • WB: BMDM, U-87 MG, C6 and RAW 264.7 whole cell lysates; Human brain, mouse heart, rat heart, mouse brain and rat brain lysates. IP: HeLa whole cell lysate. ICC/IF: HeLa cells (+/- chloroquine), HAP1 cells (+/- chloroquine) (HAP1-MAP1LC3B knockout cells used as negative cell line).
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab192890 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IP 1/50.
WB 1/2000. Detects a band of approximately 14, 16 kDa (predicted molecular weight: 15 kDa).
ICC/IF Use a concentration of 1 µg/ml.

Target

  • Function
    Probably involved in formation of autophagosomal vacuoles (autophagosomes).
  • Tissue specificity
    Most abundant in heart, brain, skeletal muscle and testis. Little expression observed in liver.
  • Sequence similarities
    Belongs to the MAP1 LC3 family.
  • Post-translational
    modifications
    The precursor molecule is cleaved by APG4B/ATG4B to form LC3-I. This is activated by APG7L/ATG7, transferred to ATG3 and conjugated to phospholipid to form LC3-II.
  • Cellular localization
    Cytoplasm > cytoskeleton. Endomembrane system. Cytoplasmic vesicle > autophagosome membrane. LC3-II binds to the autophagic membranes.
  • Information by UniProt
  • Database links
  • Alternative names
    • 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
    • MAP1ALC3 antibody
    • MAP1LC3B a antibody
    • Map1lc3b antibody
    • Microtubule associated protein 1 light chain 3 beta antibody
    • Microtubule-associated protein 1 light chain 3 beta antibody
    • Microtubule-associated proteins 1A/1B light chain 3B antibody
    • MLP3B_HUMAN antibody
    see all

Images

  • Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: LC3B knockout HAP1 cell lysate (20 µg)
    Lane 3: Human brain tissue lysate (20 µg)
    Lane 4: U87MG cell lysate (20 µg)
    Lanes 1 - 4: Merged signal (red and green). Green - ab192890 observed at 14 and 16 kDa. Red - loading control, ab8245, observed at 37 kDa.


    ab192890 was shown to specifically react with LC3B in wild-type HAP1 cells. No band was observed when LC3B knockout samples were examined. Wild-type and LC3B knockout samples were subjected to SDS-PAGE. ab192890 and ab8245 (loading control to GAPDH) were diluted 1/2000 and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

  • ab192890 staining LC3B in HeLa cells +/- Chloroquine (50μM, 24 hours). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab192890 at 1μg/ml and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor® 594), at 1/250 dilution (shown in pseudocolor red) followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • ab192890 staining LC3B in HAP1 cells (wildtype and MAP1LC3B knockout) +/- Chloroquine (50μM, 24 hours). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab192890 at 1μg/ml and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor® 594), at 1/250 dilution (shown in pseudocolor red) followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: LC3B knockout HAP1 cell lysate (20 µg)
    Lane 3: Human brain tissue lysate (20 µg)
    Lane 4: U87MG cell lysate (20 µg)
    Lanes 1 - 4: Merged signal (red and green).

    Green -target observed at 14 and 16 kDa. Red - loading control, ab8245, observed at 37 kDa.

    This western blot image is a comparison between ab192890 and a competitor's top cited rabbit polyclonal antibody.

  • LC3B was immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab192890 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab192890 at 1/1000 dilution. Veriblot for IP (HRP) (ab131366), was used as secondary antibody at 1/10000 dilution.
    Lane 1: HeLa whole cell lysate 10µg (Input).
    Lane 2: ab192890 IP in Jurkat whole cell lysate.
    Lane 3: Rabbit IgG,monoclonal [EPR25A] -Isotype Control (ab172730) instead of ab192890 in HeLa whole cell cell lysate.
    Blocking and dilution buffer and concentration: 5% NFDM/TBST.
    Exposure time: 30 seconds.

  • All lanes : Anti-LC3B antibody [EPR18709] (ab192890) at 1/2000 dilution

    Lane 1 : BMDM (Mouse bone marrow-derived macrophage cell line) whole cell lysate
    Lane 2 : BMDM (Mouse bone marrow-derived macrophage cell line) treated with 25nM rapamycin for 3 hours whole cell lysate
    Lane 3 : BMDM (Mouse bone marrow-derived macrophage cell line) treated with 5mM 3-methyl adenine for 3 hours whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 15 kDa
    Observed band size: 14,16 kDa (why is the actual band size different from the predicted?)


    Exposure time: 3 minutes


    Blocking/Dilution buffer: 5% NFDM/TBST.

  • All lanes : Anti-LC3B antibody [EPR18709] (ab192890) at 1/2000 dilution

    Lane 1 : Human brain lysate
    Lane 2 : U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate
    Lane 3 : C6 (Rat glial tumor cell line) whole cell lysate
    Lane 4 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
    Lane 5 : Mouse heart lysate
    Lane 6 : Rat heart lysate
    Lane 7 : Mouse brain lysate
    Lane 8 : Rat brain lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

    Predicted band size: 15 kDa
    Observed band size: 14,16 kDa (why is the actual band size different from the predicted?)



    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure times: Lane 1-6: 3 minutes; Lane 7 and 8: 30 seconds.

  • Immunocytochemistry/ Immunofluorescence analysis of HeLa cells labeling LC3B with ab192890 at 1/500 dilution. Cells were fixed in Methanol. Staining with ab192890 at 1/500 was carried out for 1 hour at 22°C in PBS buffer. ab150081, a Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed secondary antibody was used at 1/200 dilution. DAPI was used to counterstain.

    See Abreview

References

This product has been referenced in:
  • Liu CY  et al. LncRNA CAIF inhibits autophagy and attenuates myocardial infarction by blocking p53-mediated myocardin transcription. Nat Commun 9:29 (2018). WB ; Mouse . Read more (PubMed: 29295976) »
  • Yuan J  et al. Autophagy regulates the degeneration of the auditory cortex through the AMPK-mTOR-ULK1 signaling pathway. Int J Mol Med 41:2086-2098 (2018). Read more (PubMed: 29344647) »

See all 12 Publications for this product

Customer reviews and Q&As

Filter by Application

Filter by Species

Filter by Ratings

Application
Western blot
Sample
Human Cell lysate - whole cell (Kidney (HEK293 cells))
Gel Running Conditions
Reduced Denaturing (4-12% gel)
Loading amount
30 µg
Specification
Kidney (HEK293 cells)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Feb 15 2018

Application
Immunocytochemistry/ Immunofluorescence
Sample
African green monkey Cell (Kidney)
Permeabilization
Yes - 0.1% triton x-100
Specification
Kidney
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 37°C
Fixative
Methanol
Username

Abcam user community

Verified customer

Submitted Jan 22 2018

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Human Tissue sections (Brain)
Permeabilization
Yes - 0.3 % triton x-100
Specification
Brain
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 37°C
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Jan 19 2018

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Brain)
Permeabilization
Yes - 0.3 % triton x-100
Specification
Brain
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 37°C
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Jan 19 2018

Application
Western blot
Sample
African green monkey Cell lysate - whole cell (Kidney (COS7 cells))
Gel Running Conditions
Reduced Denaturing (4-12% gel)
Loading amount
30 µg
Specification
Kidney (COS7 cells)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Jan 12 2018

Application
Western blot
Sample
Mouse Tissue lysate - whole (Brain tissue, Neurblastoma cells)
Gel Running Conditions
Reduced Denaturing (4-12% gel)
Loading amount
30 µg
Specification
Brain tissue, Neurblastoma cells
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Jan 10 2018

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa)
Permeabilization
No
Specification
HeLa
Fixative
Methanol
Username

Dr. Kirk Mcmanus

Verified customer

Submitted Jan 16 2017

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Sign up