Cat. # | Size | Price | Inventory |
---|---|---|---|
25453S | 100 µl |
REACTIVITY | H M R |
SENSITIVITY | Endogenous |
MW (kDa) | 80 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:100 |
Flow Cytometry (Fixed/Permeabilized) | 1:400 |
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 for analysis by western immunoblot or kinase activity utilizing Protein A magnetic separation.
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.
A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein A Magnetic beads. Pre-clear enough lysate for test samples and isotype controls.
IMPORTANT: Pre-wash #73778 magnetic beads just prior to use:
Carefully remove the buffer once the solution is clear. Add 500 μl of 1X cell lysis buffer to the magnetic bead pellet, briefly vortex to wash the beads. Place tube back in magnetic separation rack. Remove buffer once solution is clear. Repeat washing step once more.
IMPORTANT: The optimal lysate concentration will depend on the expression level of the protein of interest. A starting concentration between 250 μg/ml-1.0 mg/ml is recommended.
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: To minimize masking caused by denatured IgG heavy chains (~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). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2008
revised April 2021
Protocol Id: 410
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 PKCβII 蛋白中 Val647 周围的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。
在调控分泌、基因表达、细胞增殖和肌肉收缩等多个细胞反应的级联中,蛋白激酶 C (PKC) 激活是该级联中最早期的一个活动 (1,2)。根据钙依赖性和激活因子,PKC 同工型属于三个亚家族。典型 PKC 因其 C2 结构域对钙具有依赖性,可通过其半胱氨酸富集 C1 结构域被磷脂酰丝氨酸 (PS)、甘油二酯 (DAG) 和佛波酯 (TPA, PMA) 激活。新型和非典型 PKC 均有钙依赖性,但只有新型 PKC 才能被 PS、DAG 和佛波酯激活 (3-5)。这三个 PKC 亚家族的成员包含一个假底物或自抑制结构域,可结合催化结构域中的底物结合位点,以防止在没有辅因子或激活因子的情况下被激活。PKC 活性的控制通过三个独立的磷酸化事件进行调节。活化环中的 Thr500、Thr641(自磷酸化)以及羧基末端疏水性位点 Ser660 均会发生体内磷酸化 (2)。非典型 PKC 同工型缺乏疏水区域磷酸化,这与谷氨酸的存在有关,与在更典型的 PKC 同工型中发现的丝氨酸或苏氨酸残基无关。PDK1 或相关酶负责 PKC 的激活。PKC 超家族最近新增加的成员是 PKCμ (PKD),它通过其 C1 结构域被 DAG 和 TPA 调控。PKD 的特点是有一个 PH 结构域,它有独特的底物识别和高尔基体定位特征 (6)。PKC 相关激酶 (PRK) 缺少 C1 结构域,并且不对 DAG 或佛波酯产生反应。磷脂酰肌醇脂质激活 PRK,小 Rho 家族 GTP 酶结合同源区域 1 (HR1),可调节 PRK 激酶活性 (7)。
PKCβ 有两种选择性剪切产生的同工型:PKCβI 和 PKCβII (8)。
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