LC3A Overexpression Lysate

Product Discontinued

LC3A Overexpression Lysate Summary

• Immunogen: The lysate was created in HEK293T cells, using plasmid ID RC202222 and based on accession number NM_032514. The protein contains a C-terminal DDK tag.
• Specificity: Homo sapiens microtubule-associated protein 1 light chain 3 alpha (MAP1LC3A), transcript variant 1, mRNA.
• Gene: MAP1LC3A

Applications/Dilutions

• Dilutions:
  • Western Blot
• Application Notes: This product is intended for use as a positive control in Western Blot.
  
  You will receive 1 vial of lysate (100ug), 1 vial of empty vector negative control (100ug), and 1 vial of 2xSDS sample buffer (250ul).
• Theoretical MW: 14.1 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.

Packaging, Storage & Formulations

• Storage: Store at -80C. Avoid freeze-thaw cycles.
• Buffer: RIPA buffer

Lysate Details for Array

• Type: Overexpression

Notes

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 manufactured by and sold under license from OriGene Technologies and its use is limited solely for research purposes.

Alternate Names for LC3A Overexpression Lysate

• Apg8
• APG8a
• Apg8p3
• ATG8E
• Autophagy-related protein LC3 A
• Autophagy-related ubiquitin-like modifier LC3 A
• LC3
• LC3A
• MAP1 light chain 3-like protein 1
• MAP1A/1B light chain 3 A
• MAP1A/MAP1B LC3 A
• MAP1A/MAP1B light chain 3 A
• MAP1ALC3
• MAP1BLC3
• MAP1LC3A
• Microtubule-associated protein 1 light chain 3 alpha
• microtubule-associated proteins 1A/1B light chain 3
• microtubule-associated proteins 1A/1B light chain 3A
• MLP3A

Background

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

Limitations

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.

FAQs for LC3A Lysate (NBL1-12843). (Showing 1 - 1 of 1 FAQ).

1. May we ask if it is possible to perform IF to stain LC3-I and LC3-II separately with two different fluorescent colors?
   • Yes, it is possible to perform IF stain for LC3-I and LC3-II separately with two different fluorescent colors! You will have to use two different primary antibodies and in order to avoid any potential background/cross reactivity issues, I would suggest that you employ conjugated primary antibodies for the testing. 1. Our LC3I antibody (NBP1-78964) has been designed to specifically detect the cytosolic form of the LC3 protein which is actually LC3 I (Note: LC3-II binds to the autophagic membranes). 2. There is not even a single antibody to our knowledge that would exclusively detect the LC3 II form, and you would have to detect LC3II/ autophagic membranes form with an antibody which detects LC3 I/LC3II together. Therefore you may opt second antibody from one of the followings: LC3 Antibody (NB100-2220), LC3 Antibody (NB100-2331), LC3 Antibody (NBP1-19167). All of these mentioned catalog #s come with different options for their conjugated forms and you may select appropriate conjugated forms for performing the IF staining using our explained criteria.

Bioinformatics

• Gene Symbol: MAP1LC3A