Cat. # | Size | Price | Inventory |
---|---|---|---|
3287T | 20 µl | ||
3287S | 100 µl |
REACTIVITY | H |
SENSITIVITY | Endogenous |
MW (kDa) | 258, 110, 50-80 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Simple Western™ | 1:10 - 1:50 |
Immunoprecipitation | 1:50 |
Immunohistochemistry (Paraffin) | 1:250 |
Immunofluorescence (Immunocytochemistry) | 1:200 - 1:800 |
Flow Cytometry (Fixed/Permeabilized) | 1:200 - 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
*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
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
使用与人 ROS1 蛋白中羧基末端结构域的残基相对应的一种肽,对动物进行免疫接种来产生单克隆抗体。
ROS1 是胰岛素受体家族的一种孤儿受体酪氨酸激酶,最初被发现是 UR2 肉瘤病毒蛋白的 v-ros 同源物 (1)。ROS1 由一个大的细胞外结构域组成,该结构域由六个纤连蛋白重复序列、一个跨膜结构域和一个 C 末端激酶结构域组成。作为孤儿受体,ROS1 的功能并非熟知,即便已经证明它在附睾上皮细胞分化中发挥重要作用 (2)。探索性研究最初在成胶质细胞瘤细胞中鉴定到 ROS1 的首个致癌融合蛋白 FIG-ROS1 (3),并且后续研究发现,在胆管癌 (4)、卵巢癌 (5) 和非小细胞肺癌 (NSCLC) (6) 细胞中也存在这种融合蛋白。研究人员发现,在 NSCLC 细胞中存在其他的致癌 ROS1 融合蛋白(频率约为 1.6%),其中,ROS1 激酶结构域与 CD74 和 SLC34A2 等数种不同蛋白质的氨基末端区域融合 (6-8)。ROS1 融合蛋白激活 SHP-2 磷酸酶、PI3K/Akt/mTOR、Erk 和 Stat3 通路 (3,4,9)。ROS1 激酶结构域下游有两个自磷酸化位点(Tyr2274、Tyr2334),其中任何一个都可以作为 ROS1 激酶活性的生物标志物,包括 ROS1 融合蛋白的生物标志物 (10)。
探索与本品相关的通路。
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