Cat. # | Size | Price | Inventory |
---|---|---|---|
47866S | 100 µl |
REACTIVITY | H |
SENSITIVITY | Endogenous |
MW (kDa) | 45-65 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Simple Western™ | 1:10 - 1:50 |
Immunoprecipitation | 1:50 |
Immunofluorescence (Immunocytochemistry) | 1:400 - 1:1600 |
Flow Cytometry (Fixed/Permeabilized) | 1:50 |
Flow Cytometry (Live) | 1:50 |
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
This protocol is intended for immunoprecipitation of native proteins utilizing Protein A agarose beads for analysis by western immunoblot or kinase activity.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.
NOTE: Add 1 mM PMSF (#8553) immediately prior to use.
IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 for rabbit monoclonal primary antibodies, Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal IgG1 primary antibodies, Mouse (E5Y6Q) mAb IgG2a Isotype Control #61656 for mouse monoclonal IgG2a primary antibodies, Mouse (E7Q5L) mAb IgG2b Isotype Control #53484 for mouse monoclonal IgG2b primary antibodies, and Mouse (E1D5H) mAb IgG3 Isotype Control #37988 for mouse monoclonal IgG3 primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples.
Proceed to one of the following specific set of steps.
NOTE: When using primary antibodies produced in rabbit to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) as a secondary antibody to minimize interference produced by denatured rabbit heavy chain. For proteins with a molecular weight in the range of 25 kDa, Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) is recommended to minimize interference produced by denatured mouse light chain.
When using primary antibodies produced in mouse to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Rabbit Anti-Mouse IgG (Light Chain Specific) (D3V2A) mAb (HRP Conjugate) (#58802) as a secondary antibody to minimize interference produced by denatured mouse heavy chain.
posted December 2008
revised October 2021
Protocol Id: 409
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
注:使用反渗透去离子水 (RODI) 或同等级别的水配制溶液。
注:在您的实验中加入荧光细胞染料(包括活力指示染料、DNA 染料等)时,请参考染料产品网页,了解建议的实验步骤。访问 www.cellsignal.com,了解经验证用于流式细胞术的细胞染料完整列表。
注:使用血细胞计数器或备选方法计数细胞。
注:如果使用全血,则需裂解红血细胞,并在免疫染色之前通过离心分离洗涤。
注:人 Fc 受体与兔 IgG 发生交叉反应。当感兴趣的细胞表达高水平的 Fc 受体蛋白(例如,巨噬细胞/单核细胞谱系)时,在用兔抗体进行免疫染色之前,用人 Fc 块预孵育活细胞。
注:最佳离心条件会根据细胞类型和试剂容量变动。一般,1-5 分钟 150-300g 将足以使细胞沉淀下来。
修订时间 2022 年 1 月
实验步骤编号:1865
人
使用与人 TACSTD2/TROP2 蛋白 Val90 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。
TROP2 是一种由基因 TACSTD2(肿瘤相关钙信号转导蛋白 2)编码的跨膜糖蛋白。TROP2 最初是作为一种浸润性滋养细胞的生物标记物被发现的,后来在许多类型癌细胞以及在不同发育中的各种器官与成人干细胞稳态中都有报道 (1,2)。TROP2 有一个含 EGF 甲状腺球蛋白 1 型重复序列的胞外结构域、一个跨膜结构域和一个短细胞浆结构域(其携带一个包含 PIP2 结合位点和 PKC 磷酸化位点 (Ser303) 的 HIKE 结构域)(1-4)。TROP2 通过调控多种信号转导通路而起作用,包括胞外结构域与整合素 β1 之间的相互作用(以调控 FAK 信号转导),还通过其跨膜结构域与 Claudin-1 和 Claudin-7 之间的结合(以形成紧密连接)、对胞内钙释放的调控(通过其 PIP2 结合进行调控)以及对 ERK/MAPK 通路的激活而起作用 (1,2,5-8)。所有这些功能对其在肿瘤增殖、转移和侵袭中的作用都非常重要 (1,2)。PKC 可以在 Ser303 位点磷酸化 TROP2;磷酸化改变细胞质尾构象,进一步促进其信号转导 (9)。可首先使用 TACE 通过膜内蛋白水解激活 TROP2,然后使用 Presenilin 1 和 Presenilin 2 作进一步裂解。蛋白质水解过程在肿瘤细胞增殖中不可缺少 (10,11)。
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