Cat. # | Size | Price | Inventory |
---|---|---|---|
13838S | 100 µl |
REACTIVITY | M |
SENSITIVITY | Endogenous |
MW (kDa) | 17, 14 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:200 |
Flow Cytometry (Fixed/Permeabilized) | 1:400 - 1:1600 |
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 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
小鼠
大鼠
使用与小鼠 IL-17A 蛋白 Val49 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。
IL-17(白细胞介素-17)家族由 cytokines IL-17A-F 和它们的受体(包括 IL-17RA-RE)组成 (1)。IL-17 cytokines 可由多种细胞类型产生,包括 CD4+ T 细胞的 Th17 亚型、γδ T 细胞的亚型、NK 细胞和 NKT 细胞 (2)。在对大多数 IL-17 细胞因子充分研究情况下, IL-17A 和 IL-17F 可通过诱导促炎 细胞因子、趋化因子和抗微生物肽的表达实现真菌和细菌免疫 (2)。另外,IL-17A 与一些自身免疫病的发病有关 (3)。IL-17E 能促进 Th2 细胞应答 (4)。IL-17B、IL-17C 和 IL-17D 的作用还不是很清楚,但是这些家族成员也具有诱导促炎 cytokines 的能力 (1,5,6)。IL-17 受体有一个胞外域、一个跨膜域和一个 SEFIR 域。据悉,它们通过 SEFIR 结构域来募集 SEFIR 结构域所含有的结合体 Act1,作为同源二聚体、异源二聚体或多聚体进行信号转导 (7)。与大多数 cytokines 通过 Jak/STAT 通路传递信号不同,IL-17 引起的信号转导会激活 NF-κB (8)。
IL-17A 是一种连有半胱氨酸、同型二聚体促炎性细胞因子,由 Th17 细胞(特殊的 CD4+ T 细胞系)生成(9,10)。IL-17A 可刺激促炎性 cytokines IL-1β、TNFα 和 IL-6 的生成。IL-17A 还可诱发中性粒细胞化学引诱物 IL-8、CXCL1 和 CXCL6 的生成,进而连接获得性和天然免疫 (9,10)。IL-17A 与到抵抗细菌感染的粘膜免疫紧密相关(9,11),并可能在某些自身免疫疾病中有作用(9,12)。IL-17A 主要通过结合 IL-17 其中一个受体亚基 IL-17RA 发挥作用 (13)。IL-17 结合可通过激活 Erk1/2 MAP 激酶、PI3K/Akt、p38 和 NF-κB 通路诱发 cytokines、趋化因子和其他蛋白的生成 (11,12,14)。观察了某些 Jak 和 Stat 的磷酸化现象。
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