Overview

  • Product name
  • Description
    Rabbit polyclonal to LC3B
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC/IF, IP, IHC-P, WB, IHC-Frmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Dog, Human, Zebrafish, Syrian hamster, Bacteria
    Predicted to work with: Cow
  • Immunogen

    Synthetic peptide corresponding to Human LC3B (N terminal). A synthetic peptide made to an N-terminal portion of the human LC3 protein sequence (between residues 1-100).
    Database link: Q9GZQ8

  • Positive control
    • IHC-P: Mouse hypothalamic, ovary, brain tissue. WB: HeLa, NIH/3T3 and PC-12 cells. Rat liver lysate. ICC/IF: HeLa cells; Rat hepatocyte cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab48394 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
ICC/IF Use a concentration of 1 µg/ml.
IP Use at an assay dependent concentration. 20 ug per 500 ug of protein.
IHC-P 1/200 - 1/400.
WB Use a concentration of 0.5 - 2 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).

Detects bands of approximately 17 kDa (LC3-II) and 19 kDa (LC3-I) (predicted molecular weight: 15 kDa).

IHC-Fr Use at an assay dependent concentration.

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

  • Immunostaining of LC3 (green) in the arcuate hypothalamic nucleus of lean and obese mice (A; 8 weeks of a HFD or B; 16 weeks of a HFD). DAPI (blue) was used for nuclear staining.

    The brain was excised after the mice were decapitated. Each SNC was fixed in 4% paraformaldehyde and each hypothalamus was processed for paraffin embedding and sectioned into 5.0 μm sections. Samples were incubated with primary antibodies overnight and with secondary antibodies conjugated to FITC or rhodamine for 2 hours (sc2777and sc2092, respectively; Santa Cruz Biotechnology, Santa Cruz, CA). The DAPI stain was used for nuclear staining while the Leica FW 4500 B microscope captured the images. Hypothalamic areas were observed according to the landmarks in the mouse brain atlas. Analysis and documentation of the results were performed using Leica Application Suite V3.6 (Switzerland).

  • LC3B immunofluorescence in primary follicles of PD 13 ovaries. The red dots represent LC3b and DAPI (blue) indicated cell nuclei.

    For the immunofluorescence analysis, the ovaries were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 5 μm slices. After antigen retrieval, the slides were blocked with goat serum and incubated with primary antibody (rabbit anti-LC3B at 1:200) overnight at 4°C. Alexa Fluor 594 (Invitrogen) was used as the secondary antibody in immunofluorescence assays.

    Ggpps; geranylgeranyl diphosphate synthase.

     

  • Western Blot shows lysates of HeLa (human epithelial cell line from cervix adenocarcinoma) cell line and LC3B knockout HeLa cell line (KO) untreated (-) or treated (+) with 50 uM Chloroquinine for 18 hours. PVDF membrane was probed with 0.5 ug/mL ab48394 followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody. A specific band was detected for LC3B at approximately 15 kDa (as indicated) in the parental HeLa cell line, but is not detectable in the knockout HeLa cell line. GAPDH is shown as a loading control. This experiment was conducted under reducing conditions.

  • Formalin-fixed, paraffin-embedded mouse brain tissue stained for LC3B using ab48394 at 1/200 dilution in immunohistochemical analysis. The specific signal of LC3 was detected using HRP-conjugated secondary antibody with DAB reagent, and nuclei of cells were counterstained using hematoxylin. This LC3 antibody generated a low to moderate levels of cytoplasmic staining in the glial cells. The neurons depicted a moderate to strong staining for LC3 in their cytoplasm.

  • HeLa (human epithelial cell line from cervix adenocarcinoma) cells (wild type, left; LC3B knockout HeLa, right)  stained for LC3B using ab48394 (red) at 0.3 μg/ml in ICC/IF. Primary antibody was incubated for 3 hours at room temperature, followed by NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody. Counterstained with DAPI (blue).

    LC3 was detected in immersion fixed Cloroquine treated Hela cells (left) but was not detected in LC3 knockout Hela cells (right).

  • Western blot shows lysates of mouse NIH/3T3 (mouse embryo fibroblast cell line) and rat PC-12 (rat adrenal gland pheochromocytoma cell line) cell lines untreated (-) or treated (+) with Chloroquine. PVDF membrane was probed with 0.5 ug/mL rabbit anti-LC3B polyclonal Antibody (ab48394), followed by 1:2000 dilution of goat anti-rabbit IgG secondary antibody.

  • ab48394 staining LC3B in a Rat hepatocyte by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with 0.2% Triton X-100 in PBS and blocked with 1% Donkey serum in 0.1% PBST for 60 minutes at 21°C. Samples were incubated with primary antibody (1/50 in PBS + 1% BSA) for 3 hours at 22°C. An Alexa Fluor® 394-conjugated Donkey anti-rabbit IgG polyclonal was used as the secondary antibody at a dilution of 1/200.

    See Abreview

  • All lanes : Anti-LC3B antibody (ab48394) at 1/2000 dilution

    Lane 1 : Rat whole tissue lysate - Normal liver
    Lane 2 : Rat whole tissue lysate - liver treated with AEE788 at 50 mg/kg 3 times a week for 1 week
    Lane 3 : Rat whole tissue lysate - liver treated with RAD at 2.5 mg/kg daily for 1 week

    Lysates/proteins at 30 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under non-reducing conditions.

    Predicted band size: 15 kDa
    Observed band size: 17 kDa
    why is the actual band size different from the predicted?


    Exposure time: 1 minute

    See Abreview

  • ab48394 staining LC3B (LC3-II) in HeLa cells treated with calmidazolium chloride (ab120658), by ICC/IF. Increase of LC3B (LC3-II) expression correlates with increased concentration of calmidazolium chloride, as described in literature.
    The cells were incubated at 37°C for 6h in media containing different concentrations of ab120658 (calmidazolium chloride) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab48394 (1 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

References

This product has been referenced in:
  • Magalhaes J  et al. Effects of ambroxol on the autophagy-lysosome pathway and mitochondria in primary cortical neurons. Sci Rep 8:1385 (2018). WB ; Mouse . Read more (PubMed: 29362387) »
  • González CR  et al. The balance between apoptosis and autophagy regulates testis regression and recrudescence in the seasonal-breeding South American plains vizcacha, Lagostomus maximus. PLoS One 13:e0191126 (2018). Read more (PubMed: 29385162) »
See all 161 Publications for this product

Customer reviews and Q&As

1-10 of 50 Abreviews or Q&A

Application
Immunoprecipitation
Sample
Mouse Cell lysate - whole cell (BV2)
Total protein in input
100 µg
Treatment
10nM TMT for 24hrs
Immuno-precipitation step
Protein A/G
Specification
BV2

Dr. jianhai long

Verified customer

Submitted Jan 15 2018

Application
Western blot
Sample
Human Cell lysate - whole cell (LNCaP - prostate cancer cells)
Gel Running Conditions
Reduced Denaturing (15% SDS-PAGE)
Loading amount
25 µg
Specification
LNCaP - prostate cancer cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Jun 03 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (LNCaP - prostate cancer cells)
Permeabilization
Yes - 0.2% Triton X-100
Specification
LNCaP - prostate cancer cells
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 22°C
Fixative
Formaldehyde

Dr. Armen Petrosyan

Verified customer

Submitted Jun 03 2016

Application
Western blot
Sample
Rat Cell lysate - whole cell (Rat Hepatocytes)
Gel Running Conditions
Reduced Denaturing (15% SDS-PAGE)
Loading amount
20 µg
Specification
Rat Hepatocytes
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Jun 03 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Rat Cell (Rat hepatocytes)
Permeabilization
Yes - 0.2% Triton X-100
Specification
Rat hepatocytes
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 22°C
Fixative
Formaldehyde

Dr. Armen Petrosyan

Verified customer

Submitted Mar 26 2016

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Pancreatic cancer xenograft)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tris/EDTA pH 9
Permeabilization
No
Specification
Pancreatic cancer xenograft
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 15% · Temperature: 20°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Dec 18 2015

Application
Western blot
Loading amount
30 µg
Gel Running Conditions
Reduced Denaturing
Sample
Human Cell lysate - whole cell (HEK293)
Specification
HEK293
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Feb 06 2015

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
Sample
Mouse Cell (mouse heart myocyte)
Specification
mouse heart myocyte
Permeabilization
Yes - 1% TRITON-X-100
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Feb 04 2015

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
Sample
Rat Cell (h9c2)
Specification
h9c2
Permeabilization
Yes - 1% TRITON-X-100
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Feb 04 2015

Application
Western blot
Loading amount
30 µg
Gel Running Conditions
Reduced Denaturing
Sample
Chinese Hamster Cell lysate - whole cell (CHO cell)
Specification
CHO cell
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Feb 03 2015

1-10 of 50 Abreviews or Q&A

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

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