Cat. # | Size | Price | Inventory |
---|---|---|---|
12081S | 100 µl (50 tests) |
REACTIVITY | H |
SENSITIVITY | Endogenous |
MW (kDa) | |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Immunofluorescence (Immunocytochemistry) | 1:50 |
Flow Cytometry (Fixed/Permeabilized) | 1:50 |
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
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: 182
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:407
人
小鼠, 大鼠
使用与人 Syk 蛋白中 Tyr525/526 周围残基相对应的合成磷酸肽,对动物进行免疫接种来产生单克隆抗体。
Syk 是一种在造血细胞胞内信号转导中起重要作用的蛋白酪氨酸激酶 (1-3)。Syk 能够与免疫受体胞浆域的免疫受体酪氨酸激活基序 (ITAMs) 相互作用 (4)。它能偶联激活的免疫受体进行下游信号转导活动,介导各种细胞反应,包括细胞增殖、分化、吞噬 (4)。还有证据表明,Syk 在非免疫性细胞中发挥作用,研究人员认为 Syk 是人乳腺癌细胞中的一种潜在抑癌基因 (5)。Tyr323 是 Syk 中 SH2 激酶接头区域的一个负调控磷酸化位点。Tyr323 磷酸化为 Cbl 的 TKB 结构域提供一个直接结合位点 (6,7)。Syk Tyr352 参与结合 PLCγ1 (8)。Tyr525 和 Tyr526 位于 Syk 激酶结构域的活化环;人 Syk Tyr525/526(相当于小鼠 Syk Tyr519/520)磷酸化对 Syk 功能非常重要 (9)。
探索与本品相关的通路。
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