LC3B Antibody (1251D) - Azide and BSA Free

Novus Biologicals | Catalog # NBP2-80830

Recombinant Monoclonal Antibody
Novus Biologicals
100% Guarantee

Key Product Details

Validated by

Knockout/Knockdown, Biological Validation

Species Reactivity

Human, Mouse, Rat

Applications

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

Label

Unconjugated

Antibody Source

Recombinant Monoclonal Rabbit IgG Clone # 1251D expressed in HEK293

Format

Azide and BSA Free
View all LC3B Primary 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). [Uniprot: Q9GZQ8].

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) - Azide and BSA Free

Western Blot: LC3B Antibody (1251D)Azide and BSA Free [NBP2-80830]

Western Blot: LC3B Antibody (1251D)Azide and BSA Free [NBP2-80830]

Western Blot: LC3B Antibody (1251D) - Azide and BSA Free [NBP2-80830] - 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. Image from the standard format of this antibody.
Immunocytochemistry/ Immunofluorescence: LC3B Antibody (1251D) - Azide and BSA Free [NBP2-80830]

Immunocytochemistry/ Immunofluorescence: LC3B Antibody (1251D) - Azide and BSA Free [NBP2-80830]

Immunocytochemistry/Immunofluorescence: LC3B Antibody (1251D) - Azide and BSA Free [NBP2-80830] - 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. Image from the standard format of this antibody.
Immunohistochemistry-Paraffin: LC3B Antibody (1251D) - Azide and BSA Free [NBP2-80830]

Immunohistochemistry-Paraffin: LC3B Antibody (1251D) - Azide and BSA Free [NBP2-80830]

Immunohistochemistry-Paraffin: LC3B Antibody (1251D) - Azide and BSA Free [NBP2-80830] - Analysis of a FFPE (Formalin-Fixed Paraffin-Embedded) tissue section of mouse brain using anti-LC3B antibody (clone 1251D) [Catalog # NBP2-59800] at 5ug/ml concentration (1:200 dilution). The primary antibody binding to LC3 in cells was detected using HRP
Simple Western: LC3B Antibody (1251D)Azide and BSA Free [NBP2-80830]

Simple Western: LC3B Antibody (1251D)Azide and BSA Free [NBP2-80830]

Simple Western: LC3B Antibody (1251D) - Azide and BSA Free [NBP2-80830] - Lane view shows lysates of NIH-3T3 mouse embryonic fibroblast cell line untreated (-) or treated (+) with 50 uM Chloroquine for 18 hrs, loaded at 0.2 mg/ml. A specific band was detected for LC3B at approximately 17 kDa (as indicated) using 5 ug/mL of Rabbit Anti-Human LC3B Monoclonal Antibody (1251D) [Catalog# NBP2-59800]. This experiment was conducted under reducing conditions and using the 12-230 kDa separation system. Image from the standard format of this antibody.
Knockout Validated: LC3B Antibody (1251D) - Azide and BSA Free [NBP2-80830]

Western Blot: LC3B Antibody (1251D) - Azide and BSA Free [NBP2-80830]

Western Blot: LC3B Antibody (1251D) - Azide and BSA Free [NBP2-80830] - 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. Image from the standard format of this antibody.

Applications for LC3B Antibody (1251D) - Azide and 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.

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Flow Cytometry

Formulation, Preparation, and Storage

Purification

Protein A or G purified

Formulation

PBS

Format

Azide and BSA Free

Preservative

No Preservative

Concentration

1 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

Gene Symbol

MAP1LC3B

Additional LC3B Products

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

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Product Specific Notices for LC3B Antibody (1251D) - Azide and 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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Protocols

Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.

FAQs for LC3B Antibody (1251D) - Azide and BSA Free

Showing  1 - 5 of 6 FAQs Showing All

  • Q: Can you recommend a good HRP conjugated secondary antibody for this product?

    A:

    We recommend secondary antibody NB7160 for use with LC3B Antibody (NB100-2220).

  • Q: Can you recommend a positive control (like a recombinant LC3 purified protein) for LC3B antibody NB100-2220? I am using the antibody on a western blot of mouse tissue.

    A:

    We recommend NBP1-45308 which is a full length protein that can be used for WB with NB100-2220.

     

  • Q: Does this antibody recognize LC3A or LC3C too?

    A: No; this antibody is specific for LC3B and is not expected to cross-react with LC3A or LC3C.

  • Q: Hello, somewhere on the LC3B antibody page I read it is recommended to use a 0.2uM membrane for western blot?

    A: 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.

  • Q: What is the difference in localization for LC3-I and LC3-II?

    A: LC3-I is cytoplasmic, whereas LC3-II binds to the autophagic membranes.

  • Q: What is the utility of a blocking peptide? Can you recommend a blocking peptide for this LC3B antibody NB100-2220?

    A:

    If you are interested in using a blocking peptide for this antibody, we recommend NB100-2220PEP.

    NB100-2220PEP is essentially the exact immunogen of our NB100-2220 antibody and you may use it to perform a blocking experiment to show the specificity of this antibody.

    Blocking experiments show that the band that seen and blocked upon treating the antibody with blocking peptide is the specific band detected by the antibody NB100-2220. 

  • Q: Can you recommend a good HRP conjugated secondary antibody for this product?

    A:

    We recommend secondary antibody NB7160 for use with LC3B Antibody (NB100-2220).

  • Q: Can you recommend a positive control (like a recombinant LC3 purified protein) for LC3B antibody NB100-2220? I am using the antibody on a western blot of mouse tissue.

    A:

    We recommend NBP1-45308 which is a full length protein that can be used for WB with NB100-2220.

     

  • Q: Does this antibody recognize LC3A or LC3C too?

    A: No; this antibody is specific for LC3B and is not expected to cross-react with LC3A or LC3C.

  • Q: Hello, somewhere on the LC3B antibody page I read it is recommended to use a 0.2uM membrane for western blot?

    A: 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.

  • Q: What is the difference in localization for LC3-I and LC3-II?

    A: LC3-I is cytoplasmic, whereas LC3-II binds to the autophagic membranes.

  • Q: What is the utility of a blocking peptide? Can you recommend a blocking peptide for this LC3B antibody NB100-2220?

    A:

    If you are interested in using a blocking peptide for this antibody, we recommend NB100-2220PEP.

    NB100-2220PEP is essentially the exact immunogen of our NB100-2220 antibody and you may use it to perform a blocking experiment to show the specificity of this antibody.

    Blocking experiments show that the band that seen and blocked upon treating the antibody with blocking peptide is the specific band detected by the antibody NB100-2220. 

  • Q: Can you recommend a good HRP conjugated secondary antibody for this product?

    A:

    We recommend secondary antibody NB7160 for use with LC3B Antibody (NB100-2220).

  • Q: Can you recommend a positive control (like a recombinant LC3 purified protein) for LC3B antibody NB100-2220? I am using the antibody on a western blot of mouse tissue.

    A:

    We recommend NBP1-45308 which is a full length protein that can be used for WB with NB100-2220.

     

  • Q: Does this antibody recognize LC3A or LC3C too?

    A: No; this antibody is specific for LC3B and is not expected to cross-react with LC3A or LC3C.

  • Q: Hello, somewhere on the LC3B antibody page I read it is recommended to use a 0.2uM membrane for western blot?

    A: 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.

  • Q: What is the difference in localization for LC3-I and LC3-II?

    A: LC3-I is cytoplasmic, whereas LC3-II binds to the autophagic membranes.

  • Q: What is the utility of a blocking peptide? Can you recommend a blocking peptide for this LC3B antibody NB100-2220?

    A:

    If you are interested in using a blocking peptide for this antibody, we recommend NB100-2220PEP.

    NB100-2220PEP is essentially the exact immunogen of our NB100-2220 antibody and you may use it to perform a blocking experiment to show the specificity of this antibody.

    Blocking experiments show that the band that seen and blocked upon treating the antibody with blocking peptide is the specific band detected by the antibody NB100-2220. 

  • Q: Can you recommend a good HRP conjugated secondary antibody for this product?

    A:

    We recommend secondary antibody NB7160 for use with LC3B Antibody (NB100-2220).

  • Q: Can you recommend a positive control (like a recombinant LC3 purified protein) for LC3B antibody NB100-2220? I am using the antibody on a western blot of mouse tissue.

    A:

    We recommend NBP1-45308 which is a full length protein that can be used for WB with NB100-2220.

     

  • Q: Does this antibody recognize LC3A or LC3C too?

    A: No; this antibody is specific for LC3B and is not expected to cross-react with LC3A or LC3C.

  • Q: Hello, somewhere on the LC3B antibody page I read it is recommended to use a 0.2uM membrane for western blot?

    A: 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.

  • Q: What is the difference in localization for LC3-I and LC3-II?

    A: LC3-I is cytoplasmic, whereas LC3-II binds to the autophagic membranes.

  • Q: What is the utility of a blocking peptide? Can you recommend a blocking peptide for this LC3B antibody NB100-2220?

    A:

    If you are interested in using a blocking peptide for this antibody, we recommend NB100-2220PEP.

    NB100-2220PEP is essentially the exact immunogen of our NB100-2220 antibody and you may use it to perform a blocking experiment to show the specificity of this antibody.

    Blocking experiments show that the band that seen and blocked upon treating the antibody with blocking peptide is the specific band detected by the antibody NB100-2220. 

  • Q: Can you recommend a good HRP conjugated secondary antibody for this product?

    A:

    We recommend secondary antibody NB7160 for use with LC3B Antibody (NB100-2220).

  • Q: Can you recommend a positive control (like a recombinant LC3 purified protein) for LC3B antibody NB100-2220? I am using the antibody on a western blot of mouse tissue.

    A:

    We recommend NBP1-45308 which is a full length protein that can be used for WB with NB100-2220.

     

  • Q: Does this antibody recognize LC3A or LC3C too?

    A: No; this antibody is specific for LC3B and is not expected to cross-react with LC3A or LC3C.

  • Q: Hello, somewhere on the LC3B antibody page I read it is recommended to use a 0.2uM membrane for western blot?

    A: 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.

  • Q: What is the difference in localization for LC3-I and LC3-II?

    A: LC3-I is cytoplasmic, whereas LC3-II binds to the autophagic membranes.

  • Q: What is the utility of a blocking peptide? Can you recommend a blocking peptide for this LC3B antibody NB100-2220?

    A:

    If you are interested in using a blocking peptide for this antibody, we recommend NB100-2220PEP.

    NB100-2220PEP is essentially the exact immunogen of our NB100-2220 antibody and you may use it to perform a blocking experiment to show the specificity of this antibody.

    Blocking experiments show that the band that seen and blocked upon treating the antibody with blocking peptide is the specific band detected by the antibody NB100-2220. 

  • Q: Can you recommend a good HRP conjugated secondary antibody for this product?

    A:

    We recommend secondary antibody NB7160 for use with LC3B Antibody (NB100-2220).

  • Q: Can you recommend a positive control (like a recombinant LC3 purified protein) for LC3B antibody NB100-2220? I am using the antibody on a western blot of mouse tissue.

    A:

    We recommend NBP1-45308 which is a full length protein that can be used for WB with NB100-2220.

     

  • Q: Does this antibody recognize LC3A or LC3C too?

    A: No; this antibody is specific for LC3B and is not expected to cross-react with LC3A or LC3C.

  • Q: Hello, somewhere on the LC3B antibody page I read it is recommended to use a 0.2uM membrane for western blot?

    A: 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.

  • Q: What is the difference in localization for LC3-I and LC3-II?

    A: LC3-I is cytoplasmic, whereas LC3-II binds to the autophagic membranes.

  • Q: What is the utility of a blocking peptide? Can you recommend a blocking peptide for this LC3B antibody NB100-2220?

    A:

    If you are interested in using a blocking peptide for this antibody, we recommend NB100-2220PEP.

    NB100-2220PEP is essentially the exact immunogen of our NB100-2220 antibody and you may use it to perform a blocking experiment to show the specificity of this antibody.

    Blocking experiments show that the band that seen and blocked upon treating the antibody with blocking peptide is the specific band detected by the antibody NB100-2220. 

Showing  1 - 5 of 6 FAQs Showing All
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