Cat. # | Size | Price | Inventory |
---|---|---|---|
8713S | 100 µl |
REACTIVITY | H M Mk |
SENSITIVITY | Endogenous |
MW (kDa) | 150 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Simple Western™ | 1:10 - 1:50 |
Immunoprecipitation | 1:50 |
Immunohistochemistry (Paraffin) | 1:100 - 1:400 |
Immunofluorescence (Immunocytochemistry) | 1:100 - 1:200 |
Flow Cytometry (Fixed/Permeabilized) | 1:800 |
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 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.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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: 10
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
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, 猴
大鼠
使用与人 PLCγ1 蛋白中 Ser1248 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。
磷酸肌醇特异性磷脂酶 C (PLC) 在跨膜信号转导中扮演重要角色。在激素、生长因子和神经递质等胞外刺激下,PLC 水解磷脂酰肌醇 4,5-二磷酸酯 (PIP2) 产生两个第二信使:肌醇 1,4,5-三磷酸酯 (IP3) 和甘油二酯 (DAG) (1)。现已发现至少四个 PLC 家族:PLCβ、PLCγ、PLCδ 和 PLCε。磷酸化是其中一种能调节 PLC 活性的关键机制。受体和非受体酪氨酸激酶均可激活 PLCγ (2)。PLCγ 与 EGF 和 PDGF 受体形成一种复合体,导致 PLCγ 在Tyr771、783 和 1248 位点磷酸化 (3)。Syk 磷酸化Tyr783可激活 PLCγ1 的酶活性 (4)。PLCγ2 参与 B 细胞中的抗原依赖性信号转导以及血小板中的胶原蛋白依赖性信号转导。Btk 或 Lck 磷酸化 Tyr753、759、1197 和 1217位点 与 PLCγ2 活性有关 (5,6)。
现已克隆并表征了两种哺乳动物 PLCγ 同工型(γ1 和 γ2)(7,8)。与其他 PLC 家族成员一样,PLCγ1 和 PLCγ2 包含其酶活性和底物识别所必需的钙结合域 (EF 手形、C2)和脂质互作结构域(PH、EF 手形)。只有 PLCγ 同工型具有其他保守的 SH2 和 SH3 结构域,这对其作为信号转导分子和支架蛋白发挥作用非常关键。在受到生长因子刺激后,PLCγ1 被募集(通过 SH2 结构域)到多个 RTK 中细胞浆尾区内的磷酸酪氨酸残基,以作为 RTK 的一个底物并为其他参与 RTK 信号转导的蛋白提供停靠位点 (4-6,9-12)。PLCγ1 和 γ2 还可在缺乏内在酪氨酸激酶活性的受体下游被激活。多种 G 蛋白偶联受体和 T 细胞受体的下游也有这样的报道,其中 Src、Syk 和 Tec 家族的酪氨酸激酶能结合、磷酸化并激活 PLCγ(13-15 中已论述)。受体和非受体酪氨酸激酶磷酸化酪氨酸残基可增强 PLCγ1 的激活活性,从而产生第二信使。在激动剂的刺激下,PLCγ1 在 Tyr783、Tyr711 和 Tyr1253(PLCγ2 中的 Tyr753、Tyr759 和 Tyr1217)位点被磷酸化,从而增强 PI-4,5-P2 水解 (4-6,9-12)。有趣的是,近期有证据表明,不依赖于酪氨酸激酶的 PLCγ 调节在某些系统中发挥作用。例如,在 EGF 的刺激下,Akt 的脯氨酸富集区与 PLCγ1 的 SH3 结构域相互作用,从而导致与两种酶结合、PLCγ1 在Ser1248位点 磷酸化以及细胞运动增强 (16)。这些发现表明,PLCγ1 是 RTK 和 Akt 之间的一个“支架”,从而建立一个 Akt 信号转导通路与酪氨酸激酶交互作用的机制。但 Ser1248 磷酸化的机制和功能意义尚未完全清楚,因为还有研究已经证实,PKA 介导的该位点磷酸化在经 CD3 处理的 Jurkat 细胞中对 PLCγ1 酪氨酸磷酸化和磷酸酶活性具有抑制作用 (17),表明 Ser1248 可能是 PLCγ1 活性的一种变构调节分子。
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