LC3B Antibody (1251A)

Knockout Validated: LC3B Antibody (1251A) [NBP2-46892] - Western Blot analysis shows lysates of HeLa human cervical epithelial carcinoma parental cell line and LC3B knockout HeLa cell line (KO) untreated (-) or treated (+) with 50 uM Chloroquine for 18 hours. PVDF (Polyvinylidene difluoride) membrane was probed with 2.0 ug/mL of Rabbit Anti-Human/Mouse/Rat LC3B Monoclonal Antibody (1251A) [Catalog # NBP2-46892] followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog# HAF008). A specific band was detected for LC3B at a molecular weight of 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.

Knockout Validated: LC3B Antibody (1251A) [NBP2-46892] - LC3B was detected in immersion fixed Chloroquine treated Hela cells (left) but was not detected in LC3B knockout Hela cells (right) using rabbit anti-human LC3B monoclonal antibody (1251A) [Catalog #NBP2-46892] at 0.3 ug/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody (red; Catalog # NL004) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm.

Western Blot: LC3B Antibody (1251A) [NBP2-46892] - The expression of LC3B in rat tissue. This image was submitted via customer Review.

Flow (Intracellular): LC3B Antibody (1251A) [NBP2-46892] - Jurkat cells were either untreated (A) or treated with 50uM chloroquine for 24 hours (B). An intracellular stain was performed with anti-LC3B (1251A) antibody [Catalog # NBP2-46892] (blue) and a matched isotype control [Catalog # MAB1050] (orange). Cells were fixed with 4% paraformaldehyde, following fixation, cells were permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 1 ug/mL for 30 minutes at room temperature, followed by rabbit IgG APC-conjugated secondary antibody (F0111, R&D Systems).

Immunocytochemistry/Immunofluorescence: LC3B Antibody (1251A) [NBP2-46892] - HeLa cells were treated with Chlorquine for 24 hours prior to fixation, permeabilization and incubation with anti- [Catalog # NBP2-46892] and anti tubulin (NB100-690) antibodies. Image enlargement shows the accumulation of LC3 (green) on autophagosomes in response to chloroquine treatment. Tubulin staining is shown in red and DNA is counterstained with DAPI (blue).

Immunohistochemistry-Paraffin: LC3B Antibody (1251A) [NBP2-46892] - IHC (Immunohistochemical) analysis of a formalin fixed and paraffin embedded tissue section of normal mouse brain using rabbit monoclonal LC3B (1251A) antibody [Catalog # NBP2-46892] at 1:100 dilution with HRP-DAB detection. The antibody generated a weak diffused cytoplasmic staining in most of the cells but some cells, especially within empty areas on the section, showed punctate signal also which signifies the presence of autophagy in those areas.

Flow Cytometry: LC3B Antibody (1251A) [NBP2-46892] - HeLa cells untreated or treated with 50 uM chloroquine for 24h. Image from the Alexa Fluor 405 version of this antibody.

Western Blot: LC3B Antibody (1251A) [NBP2-46892] - Analysis shows lysates of HeLa human cervical epithelial carcinoma cell line untreated (-) or treated (+) with Chloroquine. PVDF membrane was probed with 0.2 ug/mL rabbit anti-LC3B monoclonal Antibody (1251A) (Catalog # NBP2-46892, Novus Biologicals), followed by 1:2000 dilution of goat anti-rabbit IgG secondary antibody. A specific band was detected for LC3B at a molecular weight of approximately 15 kDa in CQ treated NIH-3T3 and PC12 cell lines.

Western Blot: LC3B Antibody (1251A) [NBP2-46892] - Western Blot analysis using HeLa cells treated with (+) or without (-) Chloriquine (CQ). A specific band was detected for LC3B at a molecular weight of approximately 15 kDa.

Western Blot: LC3B Antibody (1251A) [NBP2-46892] - Western Blot image of monoclonal anti-LC3B Antibody (Clone 1251D) [Catalog # NBP2-46892]. HeLa and Neuro2A cells were treated with or without 50 uM chloroquine for 24 hours as indicated. Whole cell protein was then separated on a 4-15% gel by SDS-PAGE, transferred to 0.2 um PVDF membrane for 30 min and blocked in 5% non-fat milk in TBST (Tris-buffered saline, 0.1% Tween 20). The membrane was probed with 2 ug/ml anti-LC3B Antibody in 1% milk, and detected with an anti-rabbit HRP secondary antibody using chemiluminescence. Note the accumulation of LC3B II upon chloroquine treatment. Bands for LC3 were detected at a molecular weight of approximately 15 kDa in treated HeLA cells, and both treated and untreated Neuro2A cells.

Immunocytochemistry/Immunofluorescence: LC3B Antibody (1251A) [NBP2-46892] - HeLa cells were treated with 50 uM Chloroquine for 24 hour prior to fixation in 10% buffered formalin for 10 min. Cells were permeabilized in 0.1% Triton X-100 and incubated with 20 ug/ml anti- [Catalog # NBP2-46892] and 1:500 anti-tubulin [Catalog # NB100-690] for 1 h at room temperature. LC3B reactivity (green) was detected with ant-rabbit Dylight 488 and tubulin (red) with anti-mouse Dylight 550. Nuclei were counterstained with DAPI (blue).

Flow (Intracellular): LC3B Antibody (1251A) [NBP2-46892] - HeLa human cervical epithelial carcimoa cell line either treated with 50 uM chloroquine for 24 hours (filled histogram) or untreated (open histogram) was stained with Rabbit anti-Human LC3B Alexa Fluor 647 conjugated monoclonal antiobdy (1251A) [Catalog # NBP2-46892AF647]. To facilitate intracellular staining, cells were fixed and permeabilized with FlowX FoxP3 Fixation and Permeabilization Buffer Kit (FC012-NOV).

Immunoprecipitation: LC3B Antibody (1251A) [NBP2-46892] - Western blot analysis of LC3 immunoprecipitation. 10 ug of monoclonal anti-LC3B antibody (clone 1251A) [Catalog # NBP2-46892] was used to immunoprecipitate LC3 from 200 ug of total protein from HeLa cells treated with or without 50 uM chloroquine for 24 h. Antibody-protein was captured by magnetic protein A/G beads, washed and subjected to SDS-PAGE on a 4-15% gel. Protein was transferred to 0.2 um PVDF membrane and probed with 2 ug/ml anti-LC3B (NB100-2220) and detected with an anti-rabbit HRP conjugated secondary antibody using chemiluminescence. The detection of LC3 at a molecular weight of approximately 12 kDa and the antibody heavy chain (Ab Hc) at a molecular weight of approximately 50 kDa is indicated.

• Reactivity: Hu, Mu, Rt, Rt, Po, Bt, Bv, Fi, Xp, Ze
• Applications: WB, Flow, Flow-IC, ICC/IF, IHC, IHC-P, IP, KO
• Clone: 1251A
• Clonality: Monoclonal
• Host: Rabbit
• Conjugate: Unconjugated
• Concentration: 1.0 mg/ml

LC3B Antibody (1251A) Summary

• Additional Information: Recombinant Monoclonal Antibody.
• Immunogen: Recombinant monoclonal LC3B Antibody (1251A) was made to a synthetic peptide made to an N-terminal portion of the human LC3B protein sequence (between residues 1-100). [UniProt# Q9GZQ8].
• Localization: Type I form of LC3B is cytoplasmic, whereas the type II form of LC3B binds to the autophagic membranes.
• Marker: Autophagosomes
• Predicted Species: Rat (94%), Porcine (100%), Bovine (100%), Fish (100%), Zebrafish (100%), Bat (100%), Xenopus (94%). Backed by our 100% Guarantee.
• Isotype: IgG
• Clonality: Monoclonal
• Host: Rabbit
• Gene: MAP1LC3B
• Purity: Protein G purified

Applications/Dilutions

• Dilutions:
  • Western Blot
  • Flow Cytometry
  • Immunocytochemistry/Immunofluorescence 10-20 ug/ml
  • Immunohistochemistry 1:100-1:500
  • Immunohistochemistry-Paraffin 1:100-1:500
  • Immunoprecipitation 2-10 ug
  • Flow (Intracellular) 1 - 2.5 ug/mL
  • Knockout Validated
• Application Notes: In WB this LC3B recombinant monoclonal antibody detects both LC3B I and LC3B II with chloroquine treatment. With ICC autophagosome staining was observed after treatment with chloroquine.
• Theoretical MW: 14.688 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• Control:

  LC3B Overexpression Lysate
  Neuro2a Chloroquine Treated / Untreated Cell Lysate
  HeLa Chloroquine Treated / Untreated Cell Lysate

Reactivity Notes

Mouse and Rat reactivity reported from verified customer reviews.

Packaging, Storage & Formulations

• Storage: Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.
• Buffer: PBS
• Preservative: 0.02% Sodium Azide
• Concentration: 1.0 mg/ml
• Purity: Protein G purified

Alternate Names for LC3B Antibody (1251A)

• Apg8b
• ATG8F
• Autophagy-related protein LC3 B
• Autophagy-related ubiquitin-like modifier LC3 B
• lc3b autophagy marker
• LC3B monoclonal
• LC3B
• lc3-i, ii
• LC3II
• MAP1 light chain 3-like protein 2
• MAP1A/1BLC3
• MAP1A/MAP1B LC3 B
• MAP1LC3B
• MAP1LC3B-a
• microtubule associated protein 1 light chain 3 beta
• microtubule associated protein 3 b
• microtubule associated protein 3 beta
• microtubule-associated proteins 1A/1B light chain 3
• microtubule-associated proteins 1A/1B light chain 3B

Background

Autophagy (macroautophagy) is a catabolic process which targets intracellular components such as proteins and organelles for degradation. Originally described as a bulk degradation process, current research supports its selective nature (1). Selective autophagy targets specific cellular components for degradation including the endoplasmic reticulum (2) (ER-phagy), mitochondria (3) (mitophagy), peroxisomes (3) (pexophagy), ribosomes (4) (ribophagy) and bacteria (5) (xenophagy). Autophagy relies on a newly formed phagophore, a membrane structure which elongates, sequesters cellular content, and fuses to form a double membrane vesicle known as the autophagosome. Fusion of autophagosomes with lysosomes gives rise to the autophagolysosome, where cellular components are degraded by lysosome hydrolases (1).

Autophagic flux is supported by autophagy-related proteins (Atgs) initially identified in yeast (6,7). The core autophagy machinery is comprised of 17 Atg proteins that play specific roles in autophagosome formation. Among these Atg proteins, Atg8 is not only involved in autophagosome formation but also functions in cargo selection. In mammals, several Atg8 homologues have been identified including microtubule-associated protein 1 light chain 3 alpha, beta and gamma - LC3A, LC3B, and LC3C (8) respectively, as well as GABA type A receptor-associated protein (GABARAP), GABARAP-Like1, and GABARAP-Like2 (9). LC3 (predicted molecular weight 14kD) is ubiquitously expressed and undergoes posttranslational processing after synthesis. First, the cysteine protease Atg4 cleaves a carboxy terminal sequence to generate the cytosolic form LC3-I. Next, E1-like (Atg7) and E2-like (Atg3) enzymes conjugate phosphatidylethanolamine to the newly exposed carboxyterminal glycine, generating LC3-II. Finally, the Atg12-Atg5-Atg16L1 complex participates in LC3 lipidation and autophagosome formation (10). LC3B-I to LC3B-II conversion correlates with autophagosome number and is considered the best marker to monitor autophagy.

References

1. Yu, L., Chen, Y., & Tooze, S. A. (2018). Autophagy pathway: Cellular and molecular mechanisms. Autophagy. https://doi.org/10.1080/15548627.2017.1378838

2. Forrester, A., De Leonibus, C., Grumati, P., Fasana, E., Piemontese, M., Staiano, L.,... Settembre, C. (2019). A selective ER -phagy exerts procollagen quality control via a Calnexin- FAM 134B complex. The EMBO Journal. https://doi.org/10.15252/embj.201899847

3. He, X., Zhu, Y., Zhang, Y., Geng, Y., Gong, J., Geng, J.,... Zhong, H. (2019). RNF34 functions in immunity and selective mitophagy by targeting MAVS for autophagic degradation. The EMBO Journal. https://doi.org/10.15252/embj.2018100978

4. Mathai, B., Meijer, A., & Simonsen, A. (2017). Studying Autophagy in Zebrafish. Cells. https://doi.org/10.3390/cells6030021

5. Losier, T. T., Akuma, M., McKee-Muir, O. C., LeBlond, N. D., Suk, Y., Alsaadi, R. M.,... Russell, R. C. (2019). AMPK Promotes Xenophagy through Priming of Autophagic Kinases upon Detection of Bacterial Outer Membrane Vesicles. Cell Reports. https://doi.org/10.1016/j.celrep.2019.01.062

6. Nakatogawa, H., Suzuki, K., Kamada, Y., & Ohsumi, Y. (2009). Dynamics and diversity in autophagy mechanisms: Lessons from yeast. Nature Reviews Molecular Cell Biology. https://doi.org/10.1038/nrm2708

7. Tsukada, M., & Ohsumi, Y. (1993). Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Letters. https://doi.org/10.1016/0014-5793(93)80398-E

8. Wild, P., McEwan, D. G., & Dikic, I. (2014). The LC3 interactome at a glance. Journal of Cell Science. https://doi.org/10.1242/jcs.140426

9. Igloi, G. L. (2001). Cloning, expression patterns, and chromosome localization of three human and two mouse homologues of GABAA receptor-associated protein. Genomics. https://doi.org/10.1006/geno.2001.6555

10. Glick, D., Barth, S., & Macleod, K. F. (2010). Autophagy: Cellular and molecular mechanisms. Journal of Pathology. https://doi.org/10.1002/path.2697

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for LC3B Antibody (NBP2-46892)(4)

Publications using NBP2-46892

• Wang J, Lee CJ, Deci MB et al. MiR-101a loaded extracellular nanovesicles as bioactive carriers for cardiac repair Nanomedicine Apr 8 2020 [PMID: 32278100] (IHC, IHC-Fr, Mouse)
• Martinez Legaspi S, Segatori L Aggregation Behavior of Nanoparticle-Peptide Systems Affects Autophagy Endocrinology Jul 3 2019 [PMID: 31268689] (ICC/IF, Human)
• Verwey M, Nolte EM, Joubert AM,, Theron AE. Autophagy induced by a sulphamoylated estrone analogue contributes to its cytotoxic effect on breast cancer cells. Cancer Cell Int. Dec 16 2016 [PMID: 27980456] (FLOW, Human)
• Wechman SL, Rao XM, Cheng PH et al. Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection. Viruses Jun 18 2016 [PMID: 27314377] (WB)

Reviews for LC3B Antibody (NBP2-46892) (3)

Average Rating: 5

(Based on 3 reviews)

We have 3 reviews tested in 3 species: Human, Mouse, Rat.

reviewed by: Anonymous WB Rat 07/29/2017

Summary

• Application: Western Blot
• Sample Tested: Rat tissue
• Species: Rat

reviewed by: Anonymous WB Mouse 09/28/2016

Summary

• Application: Western Blot
• Sample Tested: Liver
• Species: Mouse

Blocking

• Blocking Details: -

Details

• Detection Notes: -

reviewed by: Anonymous WB Human 09/28/2016

Summary

• Application: Western Blot
• Sample Tested: bladder cell line
• Species: Human

Blocking

• Blocking Details: -

Details

• Detection Notes: -

FAQs for LC3B Antibody (NBP2-46892). (Showing 1 - 2 of 2 FAQs).

1. Hello, somewhere on the LC3B antibody page I read it is recommended to use a 0.2uM membrane for western blot?
   • Yes, because LC3B-I is about 14-15 kDa and LC3B-II is even even smaller, the protein may slip through the 0.45 uM membrane and be lost during blotting. However, there has been success using 0.45 uM membranes; a 0.2 uM membrane is something to consider if no signal is detected.
2. What should be the composition of the lysis buffer? How much protein should be loaded per lane? 
   • We load 10 ug of total protein and the lysates are prepared in RIPA buffer. In addition, the gel must be 15-20% to maximize the resolution of LC3 and the membrane should be 0.2um PVDF.

Bioinformatics

• Gene Symbol: MAP1LC3B
• Entrez:
  • 81631(Human)
  • 67443(Mouse)
  • 64862(Rat)
• Uniprot:
  • Q9GZQ8()