Cat. # | Size | Price | Inventory |
---|---|---|---|
16177S | 100 µl |
REACTIVITY | H M |
SENSITIVITY | Endogenous |
MW (kDa) | 62 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunohistochemistry (Paraffin) | 1:1600 - 1:6400 |
Immunofluorescence (Immunocytochemistry) | 1:200 - 1:800 |
Flow Cytometry (Fixed/Permeabilized) | 1:400 - 1:1600 |
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Loading of prestained molecular weight markers (#59329, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 263
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
RECOMMENDED DETECTION REAGENTS |
SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) #8114 | SignalStain® Boost IHC Detection Reagent (AP, Rabbit) #18653 |
---|---|---|
COMPATIBLE CHROMOGEN |
SignalStain® DAB Substrate Kit #8059 | SignalStain® Vibrant Red Alkaline Phosphatase Substrate Kit #76713 |
SignalStain® Vivid Purple Peroxidase Substrate Kit #96632 | SignalStain® Ultra Blue Alkaline Phosphatase Substrate Kit #12824 | |
SignalStain® Deep Black Peroxidase Substrate Kit #72986 | ||
SignalStain® Radiant Yellow Peroxidase Substrate Kit #69644 |
NOTE: Use of detection reagents other than those specified in this protocol may require further optimization of the primary antibody to account for the different sensitivities of the detection reagents.
posted February 2010
revised June 2020
Protocol Id: 283
Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our #12727 Immunofluorescence Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised November 2013
Protocol Id: 24
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised June 2020
实验步骤编号:404
人, 小鼠
大鼠
使用与人 SQSTM1/62 蛋白中 Ser349 周围的残基相对应的合成磷酸肽对动物进行免疫接种来产生单克隆抗体。
Sequestosome 1 (SQSTM1, p62) 是一种与细胞信号转导、氧化应激和自噬有关的泛素结合蛋白 (1-4)。它最初被发现是一种可结合 p56Lck SH2 结构域的蛋白 (5),并且能单独与 PKCζ 相互作用 (6,7)。SQSTM1 后来被发现与泛素相互作用,从而为多个信号转导蛋白提供支架,并诱导蛋白酶体或溶酶体降解蛋白 (8)。SQSTM1 与 TRAF6 之间的相互作用会导致 K63 连接的 TRAF6 聚泛素化以及后续的 NF-κB 通路激活 (9)。由 SQSTM1 形成的蛋白聚合物可被自噬体降解 (4,10,11)。SQSTM1 结合自噬体膜蛋白 LC3/Atg8,从而将包含 SQSTM1 的蛋白聚合物转运到自噬体 (12)。在自噬过程中,溶酶体降解自噬体会导致 SQSTM1 水平降低,相反,自噬抑制蛋白可以稳定 SQSTM1 水平。研究表明,SQSTM1 和氧化应激之间存在关联。SQSTM1 与 KEAP1 相互作用,KEAP1 是 NRF2 的一种细胞浆抑制蛋白,而 NRF2 又是一个与细胞氧化应激反应有关的关键转录因子 (3)。因此,SQSTM1 聚集会导致 NRF2 活性增强。
在氧化应激期间,SQSTM1 的Ser349位点(小鼠 Ser351)磷酸化会增强与 KEAP1 的结合,从而增强 NRF2 活性 (13)。
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