ATG5 Overexpression Lysate

Name: ATG5 Overexpression Lysate
Brand: Novus Biologicals
SKU: NBL1-07799

Novus Biologicals, part of Bio-Techne | Catalog # NBL1-07799

Submit a review

Product Datasheet / COA / SDS

100% Guarantee

Product Summary
Product Specifications
Scientific Data
Preparation & Storage
Background
Product Documents
Specific Notices

Key Product Details

Species

Human

Applications

Western Blot

Product Summary for ATG5 Overexpression Lysate

ATG5 Transient Overexpression Lysate
Expression Host: HEK293T

Plasmid: RC210563

Accession#: NM_004849

Protein Tag: C-MYC/DDK

You will receive 1 vial of lysate (100ug), 1 vial of empty vector negative control (100ug), and 1 vial of 2xSDS sample buffer (250ul). Each vial of cell lysate contains 100ug of total protein (at 1 mg/ml). The 2xSDS Sample Buffer consists of 4% SDS, 125mM Tris-HCl pH6.8, 10% Glycerol, 0.002% Bromophenol blue, 100mM DTT.

Product Specifications

Application Notes

This product is intended for use as a positive control in Western Blot. Overexpression of the target protein was confirmed using an antibody to DDK (FLAG) epitope tag (NBP1-71705) present on the protein construct.

Each vial of cell lysate contains 100ug of total protein which should be sufficient for 20-50 reactions. Depending on over-expression level, antibody affinity and detection system, some lysates can go as low as 0.1 ug per load. We recommend starting with 5ug of cell lysate. Add an equal amount of cell lysate and 2X SDS Sample buffer and boil the SDS samples for 10 minutes before loading.

TMW

32.3 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.

Type

Overexpression

Scientific Data Images for ATG5 Overexpression Lysate

Western Blot: ATG5 Overexpression Lysate [NBL1-07799]

Western Blot: ATG5 Overexpression Lysate [NBL1-07799] - (Adult Normal) [NBL1-07799] Left-Empty vector transfected control cell lysate (HEK293 cell lysate); Right -Over-expression Lysate for ATG5.

Formulation, Preparation, and Storage

Formulation

RIPA buffer

Concentration

The exact concentration of the protein of interest cannot be determined for overexpression lysates. Please contact technical support for more information.

Shipping

The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.

Storage

Store at -80C. 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

Additional ATG5 Products

Product Documents for ATG5 Overexpression Lysate

Product Datasheets

Download Product Datasheets

COA

SDS

Download SDS

Product Specific Notices for ATG5 Overexpression Lysate

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

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

Citations
Reviews