Cat. # | Size | Price | Inventory |
---|---|---|---|
8235S | 100 µl |
REACTIVITY | H M R Mk B Pg |
SENSITIVITY | Endogenous |
MW (kDa) | 38 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunohistochemistry (Paraffin) | 1:200 - 1:800 |
Immunofluorescence (Immunocytochemistry) | 1:100 - 1:200 |
Flow Cytometry (Fixed/Permeabilized) | 1:50 - 1:200 |
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, 猴, 牛 , 猪
犬 , 马
使用与人膜联蛋白 A2 的 Phe307 周围残基相对应的合成肽,对动物进行免疫接种来产生单克隆抗体。
膜连蛋白 A2 (ANXA2)(也称脂皮素 II 或依钙蛋白-1 重链)是 36 kDa 的膜联蛋白超家族成员,通过膜联蛋白重复序列以钙依赖的方式结合磷脂和其他蛋白质 (1)。膜联蛋白 A2 包含四个重复序列,并通过四个重复序列介导蛋白与蛋白、蛋白与脂质之间的相互作用 (1-4)。它与 S100A10 形成一种结构性异源四聚体,可作为肌动蛋白细胞骨架、质膜和内吞囊泡结构之间的桥梁 (5-7)。膜连蛋白 A2 最初被认定为磷脂酶 A2 的蛋白抑制剂,随后证明膜连蛋白 A2 与大量蛋白和非蛋白伴侣相互作用,包括纤丝状肌动蛋白、血影蛋白、SNARE 复合体、RNA 和病毒颗粒 (4,6,8,9)。经证明膜连蛋白 A2 还有受体样活性,可在巨噬细胞和血管内皮细胞表面检测到,并分别介导巨噬细胞激活和凝血因子 Xa 信号转导 (10-13)。细胞表面膜连蛋白 A2 上调能通过 Src 磷酸化 Tyr23 进行调节 (14-18)。有趣的是,最近已证明细胞表面膜联蛋白 A2 的表达需要 Tyr23 磷酸化,在细胞表面,膜连蛋白 A2 介导某些胰腺癌细胞的运动性、侵犯以及整体转移潜能 (19,20)。膜连蛋白 A2 经证明还能在 PKC 激活后通过多效机制在丝氨酸残基被高度磷酸化 (21-23)。如需了解膜连蛋白 A2 磷酸化位点的完整列表,请访问 www.phosphosite.org PhosphoSitePlus®。
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