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

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

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.

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

Does this antibody recognize LC3A or LC3C too?

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

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.

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

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

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

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.

Human LC3B ELISA Kit (Colorimetric) New

Name: Human LC3B ELISA Kit (Colorimetric)
Brand: Novus Biologicals
SKU: NBP3-57043

Novus Biologicals, part of Bio-Techne | Catalog # NBP3-57043

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Product Datasheet / COA

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Product Specifications
Precision
Scientific Data
Kit Contents
Preparation & Storage
Background
Product Documents
Specific Notices

Key Product Details

Sample Type & Volume Required Per Well

Serum, plasma, tissue homogenates and other biological fluids (100 uL)

Sensitivity

0.068 ng/mL (example only; lot dependent)

Assay Range

0.156 - 10 ng/mL (example only; lot dependent)

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Product Specifications

Assay Type

Sandwich ELISA

Kit Type

ELISA Kit (Colorimetric)

Reactivity

Human

Description

Assay Length: 4.5 hours

Precision

Intra-Assay Precision (Precision within an assay) %CV < 10 (example only; lot dependent)

Inter-Assay Precision (Precision between assays) %CV < 12 (example only; lot dependent)

Scientific Data Images for Human LC3B ELISA Kit (Colorimetric)

ELISA: Human LC3B ELISA Kit (Colorimetric) [NBP3-57043]

Kit Contents for Human LC3B ELISA Kit (Colorimetric)

Pre-coated 96T strip plate, Plate sealer for 96 wells, Standard, Diluent Buffer, Detection Reagent A, Detection Reagent B, TMB Substrate, Stop Solution, Wash Buffer (30 x concentrate), Instruction manual

Preparation and Storage

Stability & Storage

Storage of components varies. See protocol for specific instructions.

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 Human LC3B ELISA Kit (Colorimetric)

Product Datasheets

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COA

Product Specific Notices for Human LC3B ELISA Kit (Colorimetric)

This product is for research use only and is not approved for use in humans or in clinical diagnosis. ELISA Kits are guaranteed for 6 months from date of receipt.

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