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Key Product Details

Species Reactivity

Human, Mouse, Rat, Porcine, Alligator, Bovine, Drosophila, Fish, Guinea Pig, Primate, Xenopus, Zebrafish

Applications

ELISA, Electron Microscopy, Flow Cytometry, Immunoblotting, Immunocytochemistry/ Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Paraffin, Immunoprecipitation, Knockdown Validated, Knockout Validated, Proximity Ligation Assay, Radioimmunoassay, Simple Western, Western Blot

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free

Concentration

1.0 mg/ml

Product Summary for ATG5 Antibody - BSA Free

Immunogen

This ATG5 Antibody was made to a synthetic peptide of an N-terminal region of the human ATG5 protein (within residues 1-50) [Swiss-Prot Q9H1Y0].

Reactivity Notes

Drosophila reactivity reported in scientific literature (PMID:33221768) Fish reactivity reported in scientific literature (PMID: 26183773). Guinea Pig reactivity reported in scientific literature (PMID: 30766882). Use in Alligator reported in scientific literature (PMID:32061056).

Localization

Cytoplasm. Co-localizes with non-muscle actin.

Specificity

This is selective for the full-length and calpain cleaved isoform proteins. The short isoform is missing amino acids 1-79. The calpain cleaved form of ATG5 is missing amino acids 195-275.

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

32 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 ATG5 Antibody - BSA Free

Western Blot: ATG5 Antibody - BSA Free [NB110-53818] - ERK phosphorylation in Atg5-/-cells depends on nutrient availability. Serum-deprived Atg5-/- MEFs display decreased ERK and p90RSK phosphorylation. Immunoblots for the indicated proteins in total lysates from 2 h serum-deprived WT and Atg5-/- MEFs. The bars represent mean+/-s.e.m. *P<0.05, ***P<0.001 compared with Con; Student's t-test, n=3. Image collected and cropped by CiteAb from the following publication (http://www.nature.com/articles/ncomms3799), licensed under a CC-BY license.
Immunocytochemistry/Immunofluorescence: ATG5 Antibody - BSA Free [NB110-53818] - ERK2 utilizes kinase-docking domains to interact with ATG5-ATG12 and LC3-II.(a) Mutations in FRS on ERK2 decrease colocalization of ERK2 with ATG5-ATG12, LC3 and WIPI1. Immunofluorescence (IF) showing colocalization (depicted as white pixels by colocalization finder application') of WT-ERK2-HA, FRS ERK2 mutants (L198A-, L232A-, L198A/L232A-, Y261A-ERK2-HA) or common docking (CD) mutant (D319N-ERK2-HA) with ATG5-ATG12 (panels 1-6), LC3 (panels 7-12) or WIPI1 (panels 13-18) in EGF-treated NIH/3T3 cells. ERK2 is stained in red, and autophagy proteins are stained in green. Scale bar, 10 um. The bars represent mean+/-s.e.m. **P<0.01, ****P<0.0001 compared with WT-ERK2-transfected cells; Student's t-test, 50 cells analysed from n=2. Image collected and cropped by CiteAb from the following publication (http://www.nature.com/articles/ncomms3799), licensed under a CC-BY license.
Immunohistochemistry: ATG5 Antibody - BSA Free [NB110-53818] - Immunohistochemistry analysis of human liver hepatocytes at 2.5 ug/mL. 40X magnification.

Applications for ATG5 Antibody - BSA Free

Application
Recommended Usage

Immunocytochemistry/ Immunofluorescence

1:250

Immunohistochemistry

1:400

Immunohistochemistry-Paraffin

1:400

Immunoprecipitation

1:10 - 1:500

Simple Western

1:50

Western Blot

1:500
Please Note: Optimal dilutions of this antibody should be experimentally determined.

Reviewed Applications

Read 8 reviews rated 4.3 using NB110-53818 in the following applications:

Published Applications

Read 266 publications using NB110-53818 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 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Background: ATG5

Atg5, Autophagy related 5 or autophagy protein 5 (theoretical molecular weight 32 kDa), belongs to a group of core autophagy-related proteins first identified in yeast and later in eukaryotic cells. Atg proteins play essential roles in the process of macroautophagy. Atg5 is considered a core autophagy protein, for its role in the formation of the autophagosome, a double membrane vesicle which engulfs proteins and organelles for delivery to the lysosome and subsequent degradation (1). Atg5 participates in the process of phagophore elongation by interacting with the ubiquitin-like protein Atg12. Formation of the Atg12-Atg5 conjugate is dependent on the activities of Atg7 (E1 ubiquitin-activating like enzyme) and Atg10 (E2 ubiquitin-activating like enzyme). Non-covalent interaction between the Atg12-Atg5 conjugate and Atg16L1, allows for the formation of a large complex which associates with the nascent phagophore. The Atg16L1 complex dissociates from the autophagosome once it is fully formed (1,2).

In the context of its role in autophagy, Atg5 plays diverse physiologically relevant roles. For example, Atg5 together with Atg7 are required for adipogenesis (3). Recently, Atg5 has been implicated in the process of B-cell receptor polarization and antigen presentation (4). In addition to its role in autophagy, Atg5 is implicated in apoptotic cell death. Interaction of Atg5 with FADD (Fas-associated protein with death domain) is involved in cell death induced by IFN-gamma. A truncated form of Atg5, a 24kDa fragment, leads to cell death by interacting with Bcl-xl and inhibiting its anti-apoptotic activity (5). Other Atg5 interacting partners include interleukin-beta (IL-beta) converting enzyme and nucleotide binding oligomerization domain protein 1, which suggest that Atg5 may play other biologically relevant roles (3).

References

1. Yang, Z., & Klionsky, D. J. (2010). Mammalian autophagy: Core molecular machinery and signaling regulation. Current Opinion in Cell Biology. https://doi.org/10.1016/j.ceb.2009.11.014

2. Rubinsztein, D. C., Shpilka, T., & Elazar, Z. (2012). Mechanisms of autophagosome biogenesis. Current Biology. https://doi.org/10.1016/j.cub.2011.11.034

3. Subramani, S., & Malhotra, V. (2013). Non-autophagic roles of autophagy-related proteins. EMBO Reports. https://doi.org/10.1038/embor.2012.220

4. Arbogast, F., Arnold, J., Hammann, P., Kuhn, L., Chicher, J., Murera, D., Gros, F. (2019). ATG5 is required for B cell polarization and presentation of particulate antigens. Autophagy. https://doi.org/10.1080/15548627.2018.1516327

5. Luo, S., & Rubinsztein, D. C. (2007). Atg5 and Bcl-2 provide novel insights into the interplay between apoptosis and autophagy. Cell Death and Differentiation. https://doi.org/10.1038/sj.cdd.4402149

Long Name

ATG5 Autophagy Related 5 Homolog

Alternate Names

APG5, ASP

Gene Symbol

ATG5

Product Documents for ATG5 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 ATG5 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 ATG5 Antibody - BSA Free (NB110-53818):

[[URL:https://www.novusbio.com/products/atg5-antibody_nb110-53818]][[Caption:… Antibody]]
Protocol: Western Blot Protocol for Atg5 Antibody (NB110-53818)

Materials
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
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-Atg5 primary antibody (NB110-53818) in blocking buffer (1:500)
Methods

1. Grow cells (e.g. HeLa or Neuro2A) in vitro to semi-confluency (70-75%).
2. Rinse cells with ice-cold 1X PBS and lyse cells with sample buffer.
3. Sonicate and incubate cells for 5 minutes at 95oC.

Tip: Cells are lysed directly in sample buffer.

4. Load 10-40 ug/lane of sample on a 12% polyacrylamide gel (SDS-PAGE).
5. Transfer proteins to a PVDF membrane for 60 minutes at 100V.

Tip: For more information on Western Blotting, see our Western Blot handbook: https://images.novusbio.com/design/BR_westernblotguide_042816b.pdf
6. After transfer, rinse the membrane with dH2O and stain with Ponceau S for 1-2 minutes to confirm efficiency of protein transfer.
7. Rinse the membrane in dH2O to remove excess stain and mark the loaded lanes and molecular weight markers using a pencil.
8. Block the membrane using blocking buffer solution (5% BSA in TBST) for 1 hour at room temperature.
9. Rinse the membrane with TBST for 5 minutes.
10. Dilute anti-Atg5 primary antibody (NB110-53818) in blocking buffer (1:500) and incubate the membrane for 1 hour at room temperature.

11. Rinse the membrane with dH2O.
12. Rinse the membrane with TBST, 3 times for 10 minutes each.
13. Incubate the membrane with diluted secondary antibody, according with product's specification, (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.
14. Rinse the membrane with TBST, 3 times for 10 minutes each.
15. Apply the detection reagent of choice (e.g. BioFX Super Plus ECL) in accordance with the manufacturer's instructions.



Deparaffinization:
1. Treat slides with Xylene: 3 changes for 5 minutes each. Drain slides for 10 seconds between changes. 2. Treat slides with 100% Reagent Alcohol: 3 changes for 5 minutes each. Drain slides for 10 seconds between changes.


Quench Endogenous Peroxidase:
1 .Place slides in peroxidase quenching solution: 15-30 minutes. To Prepare 200ml of Quenching Solution: Add 3ml of 30% Hydrogen Peroxide to 200ml of Methanol. Use within 4 hours of preparation.
2. Place slides in distilled water: 2 changes for 2 minutes each.

Retrieve Epitopes:
1. Preheat Citrate Buffer. Place 200ml of Citrate Buffer Working Solution into container, cover and place into steamer. Heat to 90-96 degrees C.
2. Place rack of slides into hot Citrate Buffer for 20 minutes. Cover.
3. Carefully remove container with slides from steamer and cool on bench, uncovered, for 20 minutes.
4. Slowly add distilled water to further cool for 5 minutes.
5. Rinse slides with distilled water. 2 changes for 2 minutes each.

Immunostaining Procedure:
1. Remove each slide from rack and circle tissue section with a hydrophobic barrier pen (e.g. Liquid Blocker-Super Pap-Pen).
2. Flood slide with wash solution. Do not allow tissue sections to dry for the rest of the procedure.
3. Drain wash solution and apply 4 drops of blocking reagent to each slide and incubate for 15 minutes.
4. Drain Blocking Reagent (do not wash off the Blocking Reagent), apply 200ul of primary antibody solution to each slide, and incubate for 1 hour.
5. Wash slides with wash solution: 3 changes for 5 minutes each.
6. Drain wash solution, apply 4 drops of secondary antibody to each slide and incubate for 1 hour.
7. Wash slides with wash solution: 3 changes for 5 minutes each.
8. Drain wash solution, apply 4 drops of DAB substrate to each slide and develop for 5-10 minutes. Check development with microscope.
9. Wash slides with wash solution: 3 changes for 5 minutes each.
10. Drain wash solution, apply 4 drops of Hematoxylin to each slide and stain for 1-3 minutes. Increase time if darker counterstaining is desired.
11. Wash slides with wash solution: 2-3 changes for 2 minutes each.
12. Drain wash solution and apply 4 drops of Bluing Solution to each slide for 1-2 minutes.
13. Rinse slides in distilled water.
14. Soak slides in 70% reagent alcohol: 3 minutes with intermittent agitation.
15. Soak slides in 95% reagent alcohol: 2 changes for 3 minutes each with intermittent agitation.
16. Soak slides in 100% reagent alcohol: 3 changes for 3 minutes each with intermittent agitation. Drain slides for 10 seconds between each change.
17. Soak slides in Xylene: 3 changes for 3 minutes each with intermittent agitation. Drain slides for 10 seconds between each change.
18. Apply 2-3 drops of non-aqueous mounting media to each slide.
19. Lay slides on a flat surface to dry prior to viewing under microscope.

NOTES:
-Use treated slides (e.g. HistoBond) to ensure adherence of FFPE sections to slide.
-Prior to deparaffinization, heat slides overnight in a 60 degrees C oven.
-All steps in which Xylene is used should be performed in a fume hood.
-For Epitope Retrieval, a microwave or pressure cooker may be substituted for the steamer method. Adjust times as necessary depending on conditions.
-For the initial IHC run with a new primary antibody, test tissues with and without Epitope Retrieval. In some instances, Epitope Retrieval may not be necessary.
-200ul is the recommended maximum volume to apply to a slide for full coverage. Using more than 200ul may allow solutions to wick off the slide and create drying artifacts. For small tissue sections, less than 200ul may be used.
-5 minutes of development with DAB substrate should be sufficient. Do not develop for more than 10 minutes. If 5 minutes of development causes background staining, further dilution of the primary antibody may be necessary.
-Hematoxylin should produce a light nuclear counterstain so as not to obscure the DAB staining. Counterstain for 1-2 minutes for nuclear antigens. Counterstain for 2-3 minutes for cytoplasmic and membranous antigens. If darker counterstaining is desired, increase the time (up to 10 minutes).

FAQs for ATG5 Antibody - BSA Free

Showing  1 - 5 of 5 FAQs Showing All
  • Q: I am interested in testing your anti ATG5 (NB110-53818SS) for Immunofluorescence in MDCK cell (canine epithelial cell line). I noticed you report reactivity of the antibody against several species but not dog. Do you know if the Ab will react against canine genome? Also for IF what fixation do you recommend?

    A: The applications and species listed for any one of our antibodies are ones we have tested and validated. Canine is not listed as it has not been tested and therefore we cannot guarantee its cross reactivity. If you would be interested in testing this antibody in canine, we can recommend our Innovator's Reward. Our Innovator's Reward is offered to reward researchers for testing new species and applications with our products. Learn more about our Innovators Reward Program. Also, please use this link to view our Immunofluorescence protocol which explains the fixation method that we recommend.

  • Q: Our customer is interested in this ATG5 antibody (cat. no. NB110-53818). Would you please confirm that this antibody would recognize ATG5 alone, as well as ATG5-ATG12 conjugate?

    A: The antibody was raised against a peptide corresponding to the N-terminal of ATG5. It should recognize ATG5 alone but it appears that ATG5 is rarely found alone in the cell.

  • Q: The arrow in the data sheet points to the ATG5/ATG12 band of 56 kDa. There is also a band of 33 kDa which I guess is ATG5? Could you explain the meaning of detecting the ATG5/ATG12 conjugate in preference to ATG5?  Does the antibody cross-react with ATG12?

    A: The Western blot image you are referring to illustrates the ATG5/ATG12 conjugate, which is formed during autophagy. During autophagy ATG5 is covalently bonds ATG12 in a ubiquitous reaction. The lysate that was used was meant to illustrate the ability to detect autophagy by Western blot via ATG5/ATG12. It is possible to detect free ATG5 in cell lysates, although it is not seen in this Western blot. The lower 2 bands do not correspond to ATG5. Our ATG5 antibody does not detect ATG12, which runs at about 23 kDa.

  • Q: Does this antibody detect free ATG5 monomer in HeLa or HEK 293 cells? 

    A: This antibody is able to detect ATG5 (unconjugated form) at ~33 kDa and the ATG12-conjugated form at ~56 kDa. In the Western blot image we have on our website, we show the antibody's ability to detect the autophagy induced conjugate ATG5/ATG12.  A customer review image shows both the conjugated and unconjugated ATG5 in rat cardiomyocytes. Several  publications show this antibody working in Western blot with mouse and human samples. We are not sure if in any of the publications HeLa or HEK 293 cells specifically were used, but if the ATG5 protein is present, this product will detect it in Western blot.

  • Q: Would you please help confirm if NB110-53818 has been tested by IHC with frozen sections?

    A: NB110-53818 has been tested with paraffin-embedded tissue, but we have not validated this antibody for IHC with frozen tissue sections.

Showing  1 - 5 of 5 FAQs Showing All
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