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Key Product Details

Validated by

Knockout/Knockdown, Biological Validation

Species Reactivity

Human, Mouse, Rat, Porcine, Alligator, Avian, Bacteria, Bovine, Canine, Chicken, Chinese Hamster, Golden Syrian Hamster, Guinea Pig, Hamster, Invertebrate, Monkey, Primate, Rabbit, Zebrafish

Applications

Chromatin Immunoprecipitation (ChIP), ELISA, Flow Cytometry, Immunoblotting, Immunocytochemistry/ Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Frozen, Immunohistochemistry-Paraffin, Immunoprecipitation, Knockdown Validated, Knockout Validated, Proximity Ligation Assay, SDS-Page, Simple Western, Western Blot

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free

Concentration

1.0 mg/ml

Product Summary for LC3B Antibody - BSA Free

Immunogen

Polyclonal LC3B Antibody was made to a synthetic peptide made to an N-terminal portion of the human LC3B protein sequence (between residues 1-100). [UniProt# Q9GZQ8]

Reactivity Notes

Use in Rat reported in scientific literature (PMID:34622072). Use in Chinese Hamster reported in scientific literature (PMID:34332287). Mouse reactivity reported in scientific literature (PMID:32814898). Bovine reactivity reported in scientific literature (PMID: 24895572). Primate reactivity reported in scientific literature (PMID: 25142602). Canine reactivity reported in scientific literature (PMID: 25839646). Avian reactivity reported in scientific literature (PMID: 29546310). Hamster reactivity reported in scientific literature (PMID: 26423766). Rabbit reactivity reported in scientific literature (PMID: 26497211). The mouse detection has been reported to be weaker than the human. Immunogen displays the following percentage of sequence identity for non-tested species: Xenopus 84%. Invertebrate reactivity reported in scientific literature (PMID: 26716072 ). Monkey reactivity reported in scientific literature (PMID: 30324853). Guinea pig reactivity reported from a verified customer review. Rat reactivity reported in scientific literature (PMID: 30744518). Bacteria reactivity reported in scientific literature (PMID: 28783414). Chicken reactivity reported in scientific literature (PMID: 30649814). Porcine reactivity reported in scientific literature (PMID: 30789643). Golden Syrian hamster reactivity reported in scientific literature (PMID: 23180219). Zebrafish reactivity reported in scientific literature (PMID: 29185873). Human reactivity reported in scientific literature (PMID:33086573). Use in Alligator reported in scientific literature (PMID:32061056).

Localization

Type I form of LC3B is cytoplasmic, whereas the type II form of LC3B binds to the autophagic membranes.

Marker

Autophagosome Marker

Clonality

Polyclonal

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 - BSA Free

Knockout Validation of LC3B Antibody in HeLa and LC3B Knockout HeLa Cells in Western Blot

Knockout Validation of LC3B Antibody in HeLa and LC3B Knockout HeLa Cells in Western Blot

Lysates of HeLa parental cell line and LC3B knockout HeLa cell line (KO) untreated (-) or treated (+) with 50 uM CQ for 18 hours. PVDF (Polyvinylidene difluoride) membrane was probed with 0.5 ug/mL of Rabbit Anti-LC3B Polyclonal Antibody (Catalog # NB100-2220) 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 Validation of LC3B Antibody in HeLa and LC3B Knockout HeLa Cells in Immunocytochemistry/Immunofluorescence

Knockout Validation of LC3B Antibody in HeLa and LC3B Knockout HeLa Cells in Immunocytochemistry/Immunofluorescence

LC3B was detected in immersion fixed CQ treated HeLa cells (left) but was not detected in LC3B knockout HeLa cells (right) using rabbit anti-human LC3B polyclonal antibody (Catalog #NB100-2220) at 0.3 ug/mL for 3 hours at room temperature. Cells were stained using the NorthernLights(TM) 557-conjugated anti-Rabbit IgG Secondary Antibody (red; Catalog # NL004) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm.
Detection of LC3 in WT and KO NSC34 Cells in Western Blot.

Detection of LC3 in WT and KO NSC34 Cells in Western Blot.

Contribution of autophagy to lipid droplet formation in WT and KO NSC34 cells.(A) WT and KO NSC 34 cells were treated with (+) or without (-) bafilomycin A (bafA) under basal or HBSS starvation conditions for 4 h and blotted for p62, LC3, and GAPDH. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/32321733) licensed under a CC-BY license.

Applications for LC3B Antibody - BSA Free

Application
Recommended Usage

Immunocytochemistry/ Immunofluorescence

1:200

Immunohistochemistry

1:200 - 1:400

Immunohistochemistry-Paraffin

1:200 - 1:400

Immunoprecipitation

20 ug/500 ug of protein

Simple Western

1:50

Western Blot

0.5 - 2.0 ug/mL
Application Notes
Use in SDS-PAGE reported in scientific literature (PMID:34315875).
Please Note: Optimal dilutions of this antibody should be experimentally determined.

Reviewed Applications

Read 51 reviews rated 4.6 using NB100-2220 in the following applications:

Published Applications

Read 1636 publications using NB100-2220 in the following applications:

Formulation, Preparation, and Storage

Purification

Immunogen affinity 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 -20C.

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 western blot

Gene Symbol

MAP1LC3B

Product Documents for LC3B Antibody - BSA Free

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot number in the search box below.

Product Specific Notices for LC3B Antibody - 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

View specific protocols for LC3B Antibody - BSA Free (NB100-2220):

LC3B Antibody: https://www.novusbio.com/products/lc3b-antibody_nb100-2220
Immunohistochemistry-Paraffin Embedded Sections

Antigen Unmasking:
Bring slides to a boil in 10 mM sodium citrate buffer (pH 6.0) then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench-top for 30 minutes.

Staining:
1. Wash sections in deionized water three times for 5 minutes each.
2. Wash sections in wash buffer for 5 minutes.
3. Block each section with 100-400 ul blocking solution for 1 hour at room temperature.
4. Remove blocking solution and add 100-400 ul diluted primary antibody. Incubate overnight at 4 C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul biotinylated diluted secondary antibody. Incubate 30 minutes at room temperature.
7. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
8. Add 100-400 ul Streptavidin-HRP reagent to each section and incubate for 30 minutes at room temperature.
9. Wash sections three times in wash buffer for 5 minutes each.
10. Add 100-400 ul DAB substrate to each section and monitor staining closely.
11. As soon as the sections develop, immerse slides in deionized water.
12. Counterstain sections in hematoxylin.
13. Wash sections in deionized water two times for 5 minutes each.
14. Dehydrate sections.
15. Mount coverslips.
[[URL:https://www.novusbio.com/products/lc3b-antibody_nb100-2220]][[Caption:L… Antibody]]
Protocol: Inhibition of Autophagy and LC3B Antibody (NB100-2220) Western Blot

Materials

Chloroquine diphosphate (CQ) (10 mM) in dH2O
1X PBS
Sample buffer, 2X Laemmli buffer: 4% SDS, 5% 2-mercaptoethanol (BME), 20% glycerol, 0.004% bromophenol blue, 0.125 M Tris HCl, pH 6.8
RIPA buffer: 150 mM NaCl, 1% NP-40 or Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0, 20 mM Tris-HCl, pH 7.5
1X Running Buffer: 25 mM Tris-base, 192 mM glycine, 0.1% SDS. Adjust to pH 8.3
1X Transfer buffer (wet): 25 mM Tris-base, 192 mM glycine, 20% methanol, Adjust to pH 8.3
TBS
TBST, TBS and 0.1% Tween
Blocking solution: TBST, 5% non-fat dry milk
rabbit anti-LC3B primary antibody (NB100-2220) in blocking buffer (~2 ug/mL)

Methods

Tip: For more information on Western Blotting, see our Western Blot handbook.

1. Grow cells (e.g. HeLa or Neuro2A) in vitro to semi-confluency (70-75%).

2. Add CQ to culture dishes to a final concentration of 50 uM and incubate overnight (16 hours). Remember to include an untreated sample as a negative control.
Note: Validated autophagy inducers should be included as positive controls.

3. Rinse cells with ice-cold 1X PBS and lyse cells with sample buffer.
Note: LC3B-I and LC3B-II are sensitive to degradation, although LC3B-I is more labile. These proteins are sensitive to freeze-thaw cycles and SDS sample buffers. Fresh samples should be analyzed quickly to prevent protein degradation.

4. Sonicate and incubate cells for 5 minutes at 95oC.
Tip: Cells are lysed directly in sample buffer or may be lysed in RIPA buffer.

5. Load samples of Chloroquine-treated and -untreated cell lysates 40 ug/lane on a 4-20% polyacrylamide gradient gel (SDS-PAGE).
Tip: For detection of LC3B it is particularly important to monitor the progress of the gel as this protein is relatively small (~14kDa).

Tip: Alternatively, for non-gradient gels, use a 20% polyacrylamide gel.

6. Transfer proteins to a 0.2 um PVDF membrane for 30 minutes at 100V.

7. After transfer, rinse the membrane with dH2O and stain with Ponceau S for 1-2 minutes to confirm efficiency of protein transfer.

8. Rinse the membrane in dH2O to remove excess stain and mark the loaded lanes and molecular weight markers using a pencil.

9. Block the membrane using blocking buffer solution (5% non-fat dry milk in TBST) for 1 hour at room temperature.

10.Rinse the membrane with TBST for 5 minutes.

11.Dilute the rabbit anti-LC3B primary antibody (NB100-2220) (~2 ug/mL) in blocking buffer and incubate the membrane for 1 hour at room temperature.

12.Rinse the membrane with dH2O.

13.Rinse the membrane with TBST, 3 times for 10 minutes each.

14.Incubate the membrane with diluted secondary antibody, according with product's specifications, (e.g. anti-rabbit-IgG HRP-conjugated) in blocking buffer for 1 hour at room temperature.
Note: Tween-20 may be added to the blocking or antibody dilution buffer at a final concentration of 0.05-0.2%, provided it does not interfere with antibody-antigen binding.

15.Rinse the membrane with TBST, 3 times for 10 minutes each.

16.Apply the detection reagent of choice (e.g. BioFX Super Plus ECL) in accordance with the manufacturer's instructions.

17.Image the blot.
Tip: LC3B-I and it's lipidated form LC3B-II have different electrophoretic mobility properties, with the lipidated form moving faster in an SDS-PAGE gel, albeit its larger molecular weight. LC3B-II runs at 14-16 kDa while LC3B-I runs at 16-18kDa.

Note: This assay measures the difference in the LC3B-II signal in the presence and absence of inhibitors (e.g., lysosomotropic agents). When autophagic flux is present or induced in a system an increase in the LC3B-II signal should be observed with the inhibitor.

FAQs for LC3B Antibody - BSA Free

Showing  1 - 5 of 12 FAQs Showing All
  • Q: What are the best blocking conditions for NB100-2220?

    A: The conditions we have had the most success with are 5% milk + 1% BSA as the block. Our lab also recommends doing an overnight incubation as this is how we run our QC tests in the lab at 4 degrees C.

  • Q: Is NB100-2220 the same or different from NBP-48614?

    A: NB100-2220 is an anti-LC3 Ab. It is similar to NBP1-48614, however they are different Abs. NB100-2220 is specific for only LC3B where as NBP1-48614 is specific for only LC3A I would recommend NB100-2220 if you would like to detect LC3B as it has been in 48 published papers and it has very good reviews. NB100-2220 does detect both LCI and LCII.

  • Q: What proteins are recognized by this antibody? On the datasheet, the Specificity section states 'This antibody is specific for LC3B.' However, in the Gene Symbol is listed as MAP1LC3A and the protein sequence Q9H492 is for MAP1LC3A. Can you tell me which target the antibody recognizes?

    A: This antibody was designed to detect both LC3I and LC3II, but we have experienced through our testing that it much more routinely picks up LC3B. It will sometimes pick up both bands, but it depends on how the cells are prepared often times if they are induced to express that second LC3A band, which is why we have it named as LC3B. We have other LC3 antibodies that do pick up both bands regularly.

  • Q: What is the isotype of this antibody? I would like to find an isotype control.

    A: This is a polyclonal antibody from rabbit. The isotype of this antibody, and all rabbit antibodies, will be IgG.

  • Q: Does this antibody also recognizes LC3A and LC3C?

    A: This antibody was raised using a synthetic peptide made to an N-terminal portion of the human LC3 protein sequence as immunogen and is expected to detect LC3B protein. The immunogenic sequence employed does not have any similarity to LC3A or LC3C and hence we do not expect any cross-reactivity. This product is one of our best sellers with great customer reviews, as well as with citations in at least 160 peer reviewed publications.

  • Q: Hello,I already have the antibody, but I was wondering if you could tell me the percent identity to a specific zebrafish LC3 protein. I know it says it works in zebrafish, but we have three and the one I am interested in is the one expressed maternally--- F1QKC7. Here is the Alignment to the human protein your antibody was made. Would you just look at it quick and tell me if the 10 aa peptide is one that is highly similar (like for example aa's 66-71). http://www.uniprot.org/align/2013102541KABHU792. LC3 Antibody (NB100-2220) It is very closely related to my maternal expressed protein. I was just wanting to confirm that it is the right thing and not background staining that I am seeing.

    A: Based on the actual immunogen sequence, I am only seeing a 31% homology to the zebrafish sequence you have provided. While the homology is low, there is still a possibility that the antibody could recognize the zebrafish protein. Just for your information, the zebrafish sequence we used for validation was accession number Q7ZUD8. I can offer a few suggestions to verify specific staining in your samples: If it is possible, I would verify by western blot with your tissue. If you can validate by two applications, the staining seen is most likely specific. With the staining, I would run a secondary only or isotype control to ensure that any background staining is not coming from the secondary antibody or other interaction. If you do have access to a knock-down or knock-out, this would be the most ideal way to verify specific staining.

  • Q: We just bought the LC3 antibody (NB100-2220) from your company, and this product arrived at our lab today. Unfortunately, I just found that the label on the antibody vial showed "pAB LC3 antibody", so we are confused with the "p", what does "P"mean, usually it means"phosphorylated". So, I am wondering what does "p" mean, as the phosphorylated antibody is not we wanted. BTW, the datasheet of this product never mentioned the "phosphorylated"

    A: I am sorry for any confusion. "pAB" stands for polyclonal antibody. Our NB100-2220 is not phosphorylated.

  • Q: I want to know how the lc3 antibody (nb100-2220) was produced and purified?

    A: Of course. It is immunogen affinity purified. The immunogen is A synthetic peptide made to an N-terminal portion of the human LC3 protein sequence (between residues 1-100)

  • Q: Why are there two bands present when I use LC3B antibody NB100-2220 for a western blot of LC3B?

    A: LC3 is a soluble protein with a molecular mass of ∼17 kDa and is distributed ubiquitously in eukaryotes. It is expressed as the splice variants LC3A, LC3B, and LC3C. All LC3 isoforms undergo post-translational modifications, especially PE conjugation (lipidation) during autophagy. Upon autophagic signal, the cytosolic form of LC3 (LC3-I) is conjugated to PE to form LC3-PE conjugate (LC3-II), which is recruited to the autophagosomal membranes.  For more information, you can refer to our resource page

  • 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: 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).

Showing  1 - 5 of 12 FAQs Showing All
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