LC3B Antibody (1251D) - BSA Free

Novus Biologicals, part of Bio-Techne | Catalog # NBP2-59800

Recombinant Monoclonal Antibody.
Novus Biologicals, part of Bio-Techne
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NBP2-59800
NBP2-59800SS
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Key Product Details

Validated by

Knockout/Knockdown, Biological Validation

Species Reactivity

Human, Mouse, Rat

Applications

Validated:

Flow (Intracellular), Immunocytochemistry/ Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Paraffin, Knockout Validated, Simple Western, Western Blot

Cited:

Western Blot

Label

Unconjugated

Antibody Source

Recombinant Monoclonal Rabbit IgG Clone # 1251D

Format

BSA Free

Concentration

1.0 mg/ml

View all LC3B Antibodies »

Product Specifications

Immunogen

Recombinant monoclonal LC3B Antibody (1251D) was made to a synthetic peptide made to an N-terminal portion of the human LC3B protein sequence (between residues 1-100). .

Localization

LC3-I is cytoplasmic. LC3-II binds to the autophagic membranes.

Clonality

Monoclonal

Host

Rabbit

Isotype

IgG

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.

Scientific Data Images for LC3B Antibody (1251D) - BSA Free

Knockout Validated: LC3B Antibody (1251D) - BSA Free [NBP2-59800]

Knockout Validated: LC3B Antibody (1251D) - BSA Free [NBP2-59800]

Knockout Validated: LC3B Antibody (1251D) [NBP2-59800] - HeLa human cervical epithelial carcinoma parental cell line and LC3B knockout HeLa cell line (KO) untreated (-) or treated (+) with 50 uM Chloroquine for 18 hrs. PVDF membrane was probed with 0.1 ug/mL of Rabbit Anti-LC3B Monoclonal Antibody (1251D) [Catalog# NBP2-59800] followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog# HAF008). 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 (Catalog # AF5718) is shown as a loading control. This experiment was conducted under reducing conditions.
Western Blot: LC3B Antibody (1251D)BSA Free [NBP2-59800]

Western Blot: LC3B Antibody (1251D)BSA Free [NBP2-59800]

Western Blot: LC3B Antibody (1251D) [NBP2-59800] - Total protein from HeLa and Neuro2A cells treated with or without 50 uM chloroquine for 24 hrs was separated on a 4-15% gel by SDS-PAGE, transferred to 0.2 um PVDF (Polyvinylidene difluoride) membrane and blocked in 5% non-fat milk in TBST (Tris-buffered saline, 0.1% Tween 20). The membrane was probed with 2.0 ug/ml anti-LC3B Antibody (1251d) [Catalog # NBP2-59800] in blocking buffer and detected with an anti-rabbit HRP secondary antibody using chemiluminescence. Note the detection of LC3I and LC3II at a molecular weight of approximately 15 kDa.
Immunocytochemistry/ Immunofluorescence: LC3B Antibody (1251D) - BSA Free [NBP2-59800]

Immunocytochemistry/ Immunofluorescence: LC3B Antibody (1251D) - BSA Free [NBP2-59800]

Immunocytochemistry/Immunofluorescence: LC3B Antibody (1251D) [NBP2-59800] - HeLa cells were treated with 50uM CQ (Chloroquine) overnight, fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X TBS + 0.5% Triton X-100. The cells were incubated with anti-LC3B (1251d) [Catalog # NBP2-59800] at 5 ug/ml overnight at 4C and detected with an anti-mouse DyLight 488 (Green) at a 1:500 dilution. Actin was detected with Phalloidin 568 (Red) at a 1:200 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.
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Applications for LC3B Antibody (1251D) - BSA Free

Application
Recommended Usage

Flow (Intracellular)

1 ug/ml

Immunocytochemistry/ Immunofluorescence

5 ug/ml

Immunohistochemistry

2 - 5ug/ml

Immunohistochemistry-Paraffin

2 - 5ug/ml

Simple Western

5 ug/ml

Western Blot

1 - 2 ug/ml
Application Notes
Western blot bands are seen at ~19 kDa, representing LC3-I, and ~17 kDa, representing LC3-II.
Please Note: Optimal dilutions of this antibody should be experimentally determined.

Formulation, Preparation, and Storage

Purification

Protein A or G purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1.0 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Background: LC3B

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

Long Name

Microtubule-associated Protein 1 Light Chain 3 beta

Alternate Names

Apg8b, ATG8F, LC3II, MAP1LC3B, anti-LC3B, atg8f antibody, LC3B ihc, LC3B immunohistochemistry, LC3B immunoprecipitation, LC3B monoclonal, LC3B western blot

Gene Symbol

MAP1LC3B

Additional LC3B Products

Product Documents for LC3B Antibody (1251D) - BSA Free

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Product Specific Notices for LC3B Antibody (1251D) - BSA Free

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.

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