Cat. # | Size | Price | Inventory |
---|---|---|---|
3633T | 20 µl | ||
3633S | 100 µl |
REACTIVITY | H |
SENSITIVITY | Endogenous |
MW (kDa) | 220 (ALK), 80 (NPM-ALK), 117 (EML4-ALK v1), 86 (EML4-ALK v3) |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:2000 |
Immunoprecipitation | 1:100 |
Immunohistochemistry (Paraffin) | 1:125 - 1:500 |
Flow Cytometry (Fixed/Permeabilized) | 1:400 - 1:800 |
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
*IMPORTANT: See the protocol on product webpage for the appropriate antibody dilution.
NOTE: Do not allow slides to dry at any time during this procedure.
NOTE: This procedure describes the conditions that are recommended for the Biocare Medical Decloaking Chamber. Device-specific settings and operating instructions should be utilized for other pressure cookers.
posted October 2015
revised June 2016
Protocol Id: 764
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
使用与人 ALK 羧基末端残基相对应的重组蛋白,对动物进行免疫接种来产生单克隆抗体。
间变性淋巴瘤激酶 (ALK) 是多效生长因子 (PTN) 的一种酪氨酸激酶受体,PTN 是与胚胎脑发育有关的生长因子 (1-3)。在表达 ALK 的细胞中,PTN 诱导 ALK 和下游效因子 IRS-1、Shc、PLCγ 和 PI3 激酶发生磷酸化 (1)。ALK 最初作为一种通过转位过程产生的核磷蛋白 (NPM)-ALK 融合蛋白发现 (4)。研究人员发现,NPM-ALK 融合蛋白是一种与间变性淋巴瘤相关的结构激活且会致癌的酪氨酸激酶 (4)。研究文献表明,NPM-ALK 激活 PLCγ 可能是有丝分裂活性的关键步骤,可能与间变性淋巴瘤的发病机理有关 (5)。
针对非小细胞肺癌 (NSCLC) 细胞系的研究文献,描述了与 ALK 和棘皮动物微管相关蛋白样 4 (EML4) 的不同的 ALK 致癌融合蛋白,并且在某些肺腺癌病例中存在相应融合转录蛋白。微管相关蛋白 EML4 的短氨基末端区域能融合到 ALK 的激酶结构域 (6-8)。
研究人员发现,ALK 与其他融合伴侣(如 TRK 融合基因 (TFG) 和 KIF5B)易位,这还与 NSCLC 相关 (6,7)。尤其是,在 3-7% 的 NSCLC 样本中发现 EML4-ALK 融合蛋白 (6-14)。
探索与本品相关的通路。
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