Cat. # | Size | Price | Inventory |
---|---|---|---|
90205S | 100 µl |
REACTIVITY | M |
SENSITIVITY | Endogenous |
MW (kDa) | 90, 100 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunofluorescence (Frozen) | 1:400 |
Immunofluorescence (Immunocytochemistry) | 1:50 - 1:200 |
Flow Cytometry (Fixed/Permeabilized) | 1:50 - 1:200 |
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
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Application of traditional antigen retrieval procedures to fixed-frozen tissues may result in excessive liberation of sections from the slide. It is highly recommended that users pre-treat glass slides with a reagent that promotes tissue adherence.(e.g., HistoGrip™) while reducing retrieval temperature to 70°C.
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 October 2017
Protocol Id: 1569
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
小鼠
使用与小鼠 GPNMB 蛋白中氨基末端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。
糖蛋白非转移性基因 B (GPNMB) 是在许多类型癌中过表达的 I 型跨膜糖蛋白。GPNMB 糖蛋白参与许多生理过程,包括介导晚期黑素体向角化细胞的运输 (1)、调节成骨细胞和破骨细胞的分化和功能 (2)、刺激树突细胞成熟、促进树突细胞黏附内皮细胞 (3)、增强组织修复中自噬体与溶酶体的融合,以及调节细胞碎片的降解 (4,5)。
尽管典型 GPNMB 表达见于多种组织包括(皮肤、心、肾、肺、肝和骨骼肌)中 (3,6),研究表明 GPNMB 表达升高经常有助于多种癌中的转移性表型(7中已论述)。GPNMB 一般在正常细胞中定位至胞内区室 (1,8),但是研究人员主要在肿瘤细胞表面上找到它 (9,10)。GPNMB 的差异性定位、表达及在许多癌症类型中作为肿瘤启动子的作用令这种蛋白质成为切实可行的治疗靶标 (11)。
GPNMB 胞外结构域可以由基质金属蛋白酶剪切并从细胞表面脱落 (12)。研究鉴定脱落酶 ADAM10 作为一种负责在乳腺癌细胞表面剪切 GPNMB 胞外结构域的肽酶。脱落的 GPNMB 胞外结构域可以通过诱导内皮细胞迁移而促进血管生成 (13)。
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