LC3A Overexpression Lysate

Name: LC3A Overexpression Lysate
Brand: Novus Biologicals
SKU: NBP2-04906

Novus Biologicals, part of Bio-Techne | Catalog # NBP2-04906

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

Key Product Details

Species

Human

Applications

Western Blot

Product Summary for LC3A Overexpression Lysate

LC3A Transient Overexpression Lysate
Expression Host: HEK293T

Plasmid: RC220473

Accession#: NM_181509

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

14.3 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 LC3A Overexpression Lysate

Western Blot: LC3A Overexpression Lysate [NBP2-04906]

Western Blot: LC3A Overexpression Lysate [NBP2-04906] - Western Blot: LC3 Overexpression Lysate (Adult Normal) [NBP2-04906] Left-Empty vector transfected control cell lysate (HEK293 cell lysate); Right -Over-expression Lysate for LC3.

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: LC3A

Human Microtubule-associated Protein 1A/1B Light Chain 3A (MAP1LC3A), also called LC3A for short, is a 121 amino acid (aa) protein with a theoretic molecular weight of ~14 kDa. LC3A belongs to the LC3 subfamily of Autophagy-related 8 (Atg8) proteins, which also includes LC3B and LC3C (1). The process of autophagy is the bulk degradation of proteins and organelles. Whether these three proteins have distinct roles in autophagy remains unclear, but they do have unique subcellular expression (2). Specifically, LC3A shows perinuclear and nuclear localization (2). The Atg8 family members share a similar structure of two amino-terminal alpha-helices and a ubiquitin-like core but are unique in aa sequence (1). LC3 utilizes a ubiquitin-like conjugation system to covalently bind to phosphatidylethanolamine (PE), also called lipidation, which is mediated by a series of steps (1, 3). Briefly, unprocessed, cytosolic LC3 (pro-LC3) is cleaved by the cysteine protease Atg4 to expose a c-terminal glycine (Gly) residue (LC3-I); the Gly is activated by E1-like enzyme Atg7, transferred to E2-like enzyme Atg3, and an E3-like complex facilitates the conjugation of LC3 with PE (LC3-II), incorporating it into the phagophore membrane during autophagosome formation (1, 3). The recruitment of LC3 to the phagophore is thought to mediate membrane elongation and closure (1, 3). Additionally, LC3 plays a role in recruiting cargo (protein aggregates, pathogens, and organelles) to autophagosomes and delivery for lysosomal degradation (1).

The process of autophagy is associated with a variety of diseases including neurodegenerative diseases, neuromuscular, tumorigenesis, and viral and bacterial infections (4). LC3 is a useful marker of autophagy in both healthy and diseased cells (4). Interestingly, LC3A has two variants (v1 and v2) which differ in N-terminal sequence due to the varying transcriptional start sites (5). One particular study found that LC3Av1, but not v2 or LC3B, was silenced in various cancer cell lines due to aberrant DNA methylation and re-expression of LC3Av1 in LC3Av1-silenced cells inhibited tumor growth, where overall findings suggest a possible tumor-suppressive role (5).

Alternative names for LC3A include Apg8, APG8a, ATG8E, Autophagy-related protein LC3 A, Autophagy-related ubiquitin-like modifier LC3 A, MAP1A/1B light chain 3 A, microtubule-associated proteins 1A/1B light chain 3, and MLP3A.

References

1. Shpilka, T., Weidberg, H., Pietrokovski, S., & Elazar, Z. (2011). Atg8: an autophagy-related ubiquitin-like protein family. Genome biology. https://doi.org/10.1186/gb-2011-12-7-226

2. Koukourakis, M. I., Kalamida, D., Giatromanolaki, A., Zois, C. E., Sivridis, E., Pouliliou, S., Mitrakas, A., Gatter, K. C., & Harris, A. L. (2015). Autophagosome Proteins LC3A, LC3B and LC3C Have Distinct Subcellular Distribution Kinetics and Expression in Cancer Cell Lines. PloS one. https://doi.org/10.1371/journal.pone.0137675

3. Weidberg, H., Shvets, E., & Elazar, Z. (2011). Biogenesis and cargo selectivity of autophagosomes. Annual review of biochemistry. https://doi.org/10.1146/annurev-biochem-052709-094552

4. Tanida, I., Ueno, T., & Kominami, E. (2004). LC3 conjugation system in mammalian autophagy. The international journal of biochemistry & cell biology. https://doi.org/10.1016/j.biocel.2004.05.009

5. Schaaf, M. B., Keulers, T. G., Vooijs, M. A., & Rouschop, K. M. (2016). LC3/GABARAP family proteins: autophagy-(un)related functions. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. https://doi.org/10.1096/fj.201600698R

Long Name

Microtubule-associated Protein 1 Light Chain 3 alpha

Alternate Names

Apg8, APG8a, Apg8p3, ATG8E, LC3, MAP1ALC3, MAP1LC3A, MLP3A

Gene Symbol

MAP1LC3A

Additional LC3A Products

Product Documents for LC3A Overexpression Lysate

Product Datasheets

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COA

SDS

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Product Specific Notices for LC3A 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.

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