Product Datasheet
LC3A Antibody
NBP1-19167
Unit Size: 0.1 ml
Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Reviews: 2 Publications: 19
Protocols, Publications, Related Products, Reviews, Research Tools and Images at:www.novusbio.com/NBP1-19167
Updated 10/23/2023 v.20.1

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Page 1 of 8 v.20.1 Updated 10/23/2023

NBP1-19167
LC3A AntibodyProduct Information
Unit Size

0.1 mlConcentration

1.0 mg/ml

Storage
Clonality

Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thawcycles.
Polyclonal

Preservative

0.02% Sodium Azide

Isotype

IgG

PurityImmunogen affinity purified

BufferPBS

Target Molecular Weight

14 kDaProduct Description
HostRabbit

Gene ID

84557

Gene Symbol

MAP1LC3A

Species

Human, Mouse, Rat, Bovine, Zebrafish

MarkerAutophagosome Marker

Specificity/Sensitivity

Although specificity between this LC3A Antibody and LC3B has not been tested,
this antibody was created to a peptide that has 100% identity to LC3A and 62%identity to LC3B.
Partial recombinant protein made to an N-terminal portion of the human LC3A
protein [Swiss-Prot# Q9H492].

ImmunogenProduct Application Details
Applications

Recommended Dilutions

Application Notes

Western Blot, Simple Western, Flow Cytometry,
Immunocytochemistry/Immunofluorescence, Immunohistochemistry,
Immunohistochemistry-Frozen, Immunohistochemistry-Paraffin
Western Blot 1:2500, Simple Western 1:40, Flow Cytometry 1:100,
Immunohistochemistry 1:100 - 1:400,
Immunocytochemistry/Immunofluorescence 1:100, ImmunohistochemistryParaffin 1:100 - 1:400, Immunohistochemistry-Frozen reported in scientific
literature (PMID 23936035)
In WB, bands are seen at approx. 14-17 kDa. In ICC/IF, autophagosome
formation has been seen in HeLa cells after treatment with 50 uM chloroquine.
In Simple Western only 10 - 15 uL of the recommended dilution is used per data
point. Separated by Size-Wes, Sally Sue/Peggy Sue.
The observed molecular weight of the protein may vary from the listed predicted
molecular weight due to post translational modifications, post translationcleavages, relative charges, and other experimental factors.

Page 2 of 8 v.20.1 Updated 10/23/2023

ImagesWestern Blot: LC3A Antibody [NBP1-19167] - Total protein from HeLaand Neuro2A cells treated with or without 50 uM chloroquine for 24hours was separated on a 4-15% gel by SDS-PAGE, transferred to 0.2
um PVDF membrane and blocked in 5% non-fat milk in TBST. The
membrane was probed with 2.0 ug/mL anti-LC3A in 1% non-fat milk inTBST and detected with an anti-rabbit HRP secondary antibody usingchemiluminescence. Note the detection LC3 II upon chloroquinetreatment.

Immunocytochemistry/Immunofluorescence: LC3A Antibody [NBP119167] - LC3/MAP1 [NBP1-19167] - LC3 antibody was tested in HeLa
cells with Dylight 488 (green). Cells were treated overnight with 50 uM
chloroquine to induce autophagosome formation. Nuclei and alphatubulin were counterstained with DAPI (blue) and Dylight 550 (red).

Immunohistochemistry-Paraffin: LC3A Antibody [NBP1-19167] - Analysisof a FFPE tissue section of mouse brain using LC3 antibody at 1:300
dilution. The signal was developed using HRP-labelled secondary
antibody and DAB reagent, and the sections/nuclei were further
counterstained with hematoxylin. Note the diffused cytoplasmic staining
of LC3 in all of the cells with highest positivity in various neurons.

Western Blot: LC3/MAP1LC3A Antibody [NBP1-19167] - Human brainlysate.

Page 3 of 8 v.20.1 Updated 10/23/2023

Immunohistochemistry-Paraffin: LC3A Antibody [NBP1-19167] - Analysis
of a FFPE tissue section of mouse liver using LC3 antibody at 1:300
dilution. The signal was developed using HRP-labelled secondary
antibody and DAB reagent, and the sections/nuclei were further
counterstained with hematoxylin. Note the diffused cytoplasmic stainingof LC3 in all of the hepatocytes and other liver cells.

Simple Western: LC3A Antibody [NBP1-19167] - Image shows a specificband for LC3 in 0.5 mg/mL of Neuro2A lysate. This experiment wasperformed under reducing conditions using the 12-230 kDa separation
system.

Page 4 of 8 v.20.1 Updated 10/23/2023

Publications
Jiang Z, Bo L, et al. Overexpression of homeodomain-interacting protein kinase 2 (HIPK2) attenuates sepsismediated liver injury by restoring autophagy. Cell Death Dis 2018-08-28 [PMID: 30154452] (WB, Mouse)
Yang A, Herter-Sprie G, Zhang H, et al. Autophagy Sustains Pancreatic Cancer Growth through Both CellAutonomous and Nonautonomous Mechanisms. Cancer Discov. 2018-01-09 [PMID: 29317452] (IF/IHC, Mouse)
Korski KI, Kubli DA, Wang BJ et al. Hypoxia Prevents Mitochondrial Dysfunction and Senescence in Human c-Kit+
Cardiac Progenitor Cells. Stem Cells 2019-01-10 [PMID: 30629785] (ICC/IF, Human)
Kohler LJ, Reed SR, Sarraf SA et al. Effector Protein Cig2 Decreases Host Tolerance of Infection by Directing
Constitutive Fusion of Autophagosomes with the Coxiella-Containing Vacuole MBio 2016 Jul 20 [PMID: 27435465](WB). Elife. 2016-07-20 [PMID: 27435465] (WB)
Details:
This citation used the PE version of this antibody.
Rao S, Tortola L, Perlot T et al. A dual role for autophagy in a murine model of lung cancer. Nat Commun. 2014-0101 [PMID: 24445999] (IHC-P, Mouse)
Saito T, Asai K, Sato S et al. Autophagic vacuoles in cardiomyocytes of dilated cardiomyopathy with initiallydecompensated heart failure predict improved prognosis Autophagy 2016-02-18 [PMID: 26890610] (IHC-P, Human)
Zois CE, Giatromanolaki A, Sivridis E et al. Autophagic flux in normal mouse tissues: focus on endogenous LC3A
processing. Autophagy 2011-11-01 [PMID: 21997374]
Yang Annan, Rajeshkumar N V, Wang Xiaoxu et al. Autophagy is critical for pancreatic tumor growth and progression
in tumors with p53 alterations. Cancer Discov. 2014-05-29 [PMID: 24875860]
Campos T, Ziehe J, Palma M et al. Rheb promotes cancer cell survival through p27Kip1-dependent activation ofautophagy Mol. Carcinog. 2015-01-15 [PMID: 25594310] (WB, Human)
Giovannini M, Bonne NX, Vitte J et al. mTORC1 inhibition delays growth of neurofibromatosis type 2 schwannoma.
Neuro-oncology 2014-01-10 [PMID: 24414536] (IHC-P, Mouse)
Bruntz RC, Taylor HE, Lindsley CW, Brown HA. Phospholipase D2 mediates survival signaling through direct
regulation of Akt in glioblastoma cells. J Biol Chem. 2013-11-20 [PMID: 24257753] (WB, Human)
Howell GM, Gomez H, Collage RD et al. Augmenting Autophagy to Treat Acute Kidney Injury during Endotoxemia inMice. PLoS One 2013-07-30 [PMID: 23936035] (ICC/IF, IHC-Fr, Mouse)
More publications at http://www.novusbio.com/NBP1-19167

Page 5 of 8 v.20.1 Updated 10/23/2023

Procedures
Western Blot protocol specific for LC3 Antibody (NBP1-19167)[[URL:https://www.novusbio.com/products/lc3a-antibody_nbp1-19167]][[Caption:LC3A Antibody]]
Protocol: Inhibition of Autophagy and LC3 Antibody (NBP1-19167) 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, pH8.0, 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
TBSTBST, TBS and 0.1% Tween
Blocking solution: TBST, 5% non-fat dry milk
rabbit anti-LC3 primary antibody (NBP1-19167) 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 toinclude 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: LC3-I and LC3-II are sensitive to degradation, although LC3-I is more labile. These proteins are sensitive tofreeze-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 gradientgel (SDS-PAGE).
Tip: For detection of LC3 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 ofprotein 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-LC3 primary antibody (NBP1-19167) (~2 ug/mL) in blocking buffer and incubate thePage 6 of 8 v.20.1 Updated 10/23/2023

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-rabbitIgG 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'sinstructions.
17.Image the blot.
Tip: LC3-I and it's lipidated form LC3-II have different electrophoretic mobility properties, with the lipidated form
moving faster in an SDS-PAGE gel, albeit its larger molecular weight. LC3-II runs at 14-16 kDa while LC3-I runs at 16-18kDa.
Note: This assay measures the difference in the LC3-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 LC3-II signal
should be observed with the inhibitor.
Immunohistochemistry-Paraffin protocol for LC3A Antibody (NBP1-19167)[[URL:https://www.novusbio.com/products/lc3a-antibody_nbp1-19167]][[Caption:LC3A Antibody]]
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 dH2O three times for 5 minutes each.
2. Wash section in wash buffer (1X PBS/0.1% Tween-20 (1X PBST)) for 5 minutes.
3. Block each section with 100-400 ul blocking solution (1X PBST, 5% goat serum) for 1 hour at room temperature.
4. Remove blocking solution and add 100-400 ul primary antibody diluted in 1X PBST, 5% goat serum to eachsection. Incubate overnight at 4C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul biotinylated secondary antibody, diluted in 1X PBST, 5% goat serum. 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 Striptavidin-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 dH2O.
12. Counterstain sections in hematoxylin.
13. Wash sections in dH2O two times for 5 minutes each.
14. Dehydrate sections.
15. Mount coverslips.

Page 7 of 8 v.20.1 Updated 10/23/2023

Immunocytochemistry/Immunofluorescence Protocol for LC3 Antibody (NBP1-19167)[[URL: https://www.novusbio.com/products/lc3a-antibody_nbp1-19167]][[Caption:LC3A Antibody]]
Immunocytochemistry Protocol
Culture cells to appropriate density in 35 mm culture dishes or 6-well plates.
1. Remove culture medium and add 10% formalin to the dish. Fix at room temperature for 30 minutes.
2. Remove the formalin and add ice cold methanol. Incubate for 5-10 minutes.
3. Remove methanol and add washing solution (i.e. PBS). Be sure to not let the specimen dry out. Wash three timesfor 10 minutes.
4. To block nonspecific antibody binding incubate in 10% normal goat serum from 1 hour to overnight at room
temperature.
5. Add primary antibody at appropriate dilution and incubate at room temperature from 2 hours to overnight at room
temperature.
6. Remove primary antibody and replace with washing solution. Wash three times for 10 minutes.
7. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
8. Remove antibody and replace with wash solution, then wash for 10 minutes. Add Hoechst 33258 to wash solution
at 1:25,0000 and incubate for 10 minutes. Wash a third time for 10 minutes.
9. Cells can be viewed directly after washing. The plates can also be stored in PBS containing Azide covered inParafilm (TM). Cells can also be cover-slipped using Fluoromount, with appropriate sealing.
*The above information is only intended as a guide. The researcher should determine what protocol best meets theirneeds. Please follow safe laboratory procedures.

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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.
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