货号：17-10232

品牌：Merck Millipore

规格：

目录价：￥5153.00

市场价格：￥4380.05

会员价格：￥4122.40

金山科研平台，产品价格货期咨询微信：jinshanbio Description: LC3-II Enrichment Kit (Western Blot) View All» Qty/Pk: 15 assays (1 kit) View All» Product Overview: Autophagy, a degradative pathway that provides recycled nutrients to cells under stress, plays both protective and deleterious roles in many diseases, including cancer, neurodegeneration, and infections. Members of the LC3 family play a key role in the maturation of the autophagosome, the central organelle of autophagy. LC3 precursors are proteolytically processed to form LC3-I, which is diffusely distributed in the cytosol. Upon initiation of autophagy, the C-terminal glycine of LC3-I is modified by addition of a phosphatidylethanolamine (PE) to form LC3-II, which translocates rapidly to nascent autophagosomes in a punctate distribution. Given the pronounced changes in biochemical properties and subcellular localization of LC3 upon initiation of autophagy, detection of LC3 by various means has become the gold standard method for estimating autophagosome quantity within the cell. However, detecting and interpreting the relative amounts of LC3-I and LC3-II in standard assay methods can be complicated. When LC3 is immunochemically detected by Western Blotting following SDS-polyacrylamide gel electrophoresis, LC3-II typically appears as a slightly lower band than the 18 kDa LC3-I band as a result of the greater hydrophobicity of LC3-II. However, in some instances, the LC3-I and LC3-II bands are incompletely resolved, which complicates analysis. Also, even when LC3-I and LC3-II are adequately separated on an immunoblot, changes in the relative ratios of the isoforms may not be directly proportional to autophagosome quantity, due to differential immunoreactivity of the isoforms. Finally, there is confusion about whether the best indicator of autophagy is 1) absolute quantity of LC3-II, 2) the relative ratio of LC3-II to LC3-I alone, or 3) the relative ratio of LC3-I to the sum of LC3-I and LC3-II. A review solely devoted to interpretation of LC3 immunoblotting concludes that the absolute quantity of LC3-II is the best indicator of autophagy. EMD Millipore’s LC3-II Enrichment Kit (Western Blot) enables sensitive and accurate quantification of autophagosome density by utilizing a selective permeabilization procedure that removes cytosolic LC3-I and retains autophagosome-bound LC3-II. This procedure reveals quantities of LC3-II, without interference from LC3-I, by Western blotting analysis. The kit also includes a lysosomal inhibitor, to prevent degradation of LC3-II that occurs upon fusion of the autophagosome with the lysosome. In addition, an antibody against a mitochondrial outer membrane protein TOMM22 is included for use as a control for specificity of permeabilization, to ensure that organelles remain intact after treatment. View All» Key Applications: Western Blotting View All» Application Notes: EMD Millipore’s LC3-II Enrichment Kit (Western Blot) enables sensitive and accurate quantification of autophagosome density by utilizing a selective permeabilization procedure that removes cytosolic LC3-I and retains autophagosome-bound LC3-II. This procedure reveals quantities of LC3-II, without interference from LC3-I, by Western blotting analysis. The kit also includes a lysosomal inhibitor, to prevent degradation of LC3-II that occurs upon fusion of the autophagosome with the lysosome. In addition, an antibody against a mitochondrial outer membrane protein TOMM22 is included for use as a control for specificity of permeabilization, to ensure that organelles remain intact after treatment. View All» Usage Statement: Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals. View All» Components: LC3-II Enrichment Component Box (Store at 2-8º C)1. Autophagy Reagent A: One vial, lyophilized. 2. 10x Enrichment Buffer: One bottle containing 10 mL 10x Enrichment Buffer. 3. 5x Assay Buffer: One bottle containing 15 mL 5x Assay Buffer.4. 10x RIPA Lysis Buffer: One bottle containing 1 mL 10x RIPA Lysis Buffer.Western Blot Detection Component Box (Store at -20ºC)1. Protease Inhibitor Cocktail: One vial containing 20 µL of 1000x Protease Inhibitor Cocktail. 2. Anti-LC3 antibody: One vial containing 20 µL of 1000x anti-LC3 rabbit monoclonal antibody. 3. Anti-TOMM22 antibody: One vial containing 20 µL of 1000x anti-TOMM22 mouse monoclonal antibody. 4. Goat anti-Mouse IgG HRP, Secondary Antibody: One vial containing 30 µL of 2000X goat anti-mouse HRP conjugate.5. Goat anti-Rabbit IgG HRP, Secondary Antibody: One vial containing 30 µL of 2000X goat anti-rabbit HRP conjugate. View All» Brand Family: Chemicon View All» Storage Conditions: Maintain the unopened kit boxes at the indicated temperatures. Upon receipt, store 10x RIPA Lysis Buffer at room temperature.The kit may be used for up to 4 months after receipt. Avoid repeated freeze/thaw cycles, and aliquot if necessary. View All» Product Name: LC3-II Enrichment Kit (Western Blot) View All» Materials Required but Not Delivered: 1. Pipettors and tips capable of accurately measuring 1-1000 L2. Graduated serological pipettes3. Microcentrifuge tubes 4. Mechanical vortex5. Rocker6. Distilled or deionized water7. 1x Dulbecco’s Phosphate Buffered Saline (D-PBS) 8. 1x Tris-buffered saline containing 0.05% Tween-20 (TBST)9. 3% Non-fat dry milk diluted in TBST (TBST-MILK)10. Earle’s Balanced Salt Solution (EBSS) View All»