Cat. # | Size | Price | Inventory |
---|---|---|---|
13113T | 20 µl | ||
13113S | 100 µl |
REACTIVITY | H |
SENSITIVITY | Endogenous |
MW (kDa) | 90 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:100 |
Immunofluorescence (Immunocytochemistry) | 1:50 - 1:200 |
Flow Cytometry (Fixed/Permeabilized) | 1:100 - 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 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 equivalently purified water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multiwell plates, chamber slides or on coverslips.
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 December 2010
Protocol Id: 32
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
通过采用与人 CD71 蛋白的 Leu146 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。
转铁蛋白受体 1(CD71、TFRC)是负责通过受体介导的内吞作用摄取细胞铁的 II 型跨膜受体和载体蛋白 (1)。细胞表面的中性 pH 促进铁结合糖蛋白转铁蛋白 (Tf) 与 CD71 受体结合。受体-配体复合体通过受体介导的内吞作用进入细胞并内化至内体中。相对较低的内体 pH 导致铁-转铁蛋白结合位点周围的局部电荷环境变化并导致铁释放 (2)。受体-配体复合体再循环至细胞表面,在这里,转铁蛋白与 CD71 受体解离 (2)。普遍表达的转铁蛋白受体连续地再循环并接受笼形蛋白-介导的内吞,无论配体结合状态是什么。已经充分研究受体和配体之间的相互作用。CD71 的螺旋结构域与转铁蛋白 C 臂直接相互作用并且在 Tf 中诱导构象变化以促进转运过程 (3)。受体 CD71 和转铁蛋白之间的相互作用由血色素沉着病膜蛋白 (HFE) 介导。HFE 结合 CD71 的 α-螺旋结构域,阻断 CD71-转铁蛋白复合体的形成并抑制铁摄取 (4,5)。除结合转铁蛋白之外,CD71 还与 H-铁蛋白在细胞表面相互作用并且将这种胞内铁贮藏蛋白转运至细胞内体和溶酶体 (6)。其他研究表明,转铁蛋白受体是许多沙粒病毒和至少一种逆转录病毒的进化上保守的受体 (7,8)。CD71 异常表达见于多种癌中,包括甲状腺癌、淋巴瘤和 T 系白血病,从而提示定向抑制转铁蛋白受体可能具有治疗性作用 (9,10)。
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