Cat. # | Size | Price | Inventory |
---|---|---|---|
2348T | 20 µl | ||
2348S | 100 µl | ||
2348L | 300 µl |
REACTIVITY | H M R Mk |
SENSITIVITY | Endogenous |
MW (kDa) | 56 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunofluorescence (Immunocytochemistry) | 1:50 |
Flow Cytometry (Fixed/Permeabilized) | 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
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, 小鼠, 大鼠, 猴
使用与人 Chk1 蛋白的 Ser345 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。
Chk1 激酶在 ATM/ATR 激酶下游发挥作用并且在 DNA 损伤检查点控制、胚胎发育和肿瘤抑制中起着重要作用 (1)。Chk1 的激活需要在 Ser317 和 Ser345 位点由 ATM/ATR 引起的磷酸化,随后在Ser296 发生自磷酸化。该激活会出现以响应 DNA 复制受阻和某些形式的遗传毒性应激 (2)。在检查点激活后 Ser345 位点的磷酸化促使 Chk1 定位至胞核 (3),而Ser317 位点的磷酸化以及 PTEN 的位点特异性磷酸化则使其在 DNA 复制停顿后可以再进入细胞周期 (4)。Chk1 对细胞周期发挥其检查点机制,在某种程度上是通过调控 cdc25 磷酸酶家族发挥的。cdc25A 的 Chk1 磷酸化导引其供蛋白酶解并且通过 14-3-3 结合作用抑制其活性 (5)。激活的 Chk1 可以通过 Ser216 位点的磷酸化使 cdc25C 失活,从而阻断激活 cdc2 及转变成有丝分裂 (6)。据已表明,中心体 Chk1 可使 cdc25B 磷酸化并抑制其激活 CDK1-细胞周期蛋白 B1,从而终止有丝分裂纺锤体的形成和染色质的凝缩 (7)。另外,Chk1 在纺锤体检查点功能中通过调控 aurora B 和 BubR1 而发挥作用 (8)。调查研究表明,Chk1 可作为癌症治疗的药物靶标,这是由于在许多癌细胞系中对 Chk1 的抑制会导致细胞死亡 (9)。
探索与本品相关的通路。
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