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REACTIVITY | H |
SENSITIVITY | Endogenous |
MW (kDa) | |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Immunofluorescence (Immunocytochemistry) | 1:50 - 1:200 |
Flow Cytometry (Fixed/Permeabilized) | 1:100 |
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
使用与人 IRF-5 蛋白中 Gly480 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。
干扰素调节因子 (IRF) 包括一个转录因子家族,这些转录因子在 Jak/Stat 通路内部发挥作用以调节响应于病毒性感染的干扰素 (IFN) 和 IFN 诱导型基因表达 (1)。IRF 在病原体防御、自身免疫性、淋巴细胞发育、细胞生长和转化易发i性方面发挥重要作用。IRF 家族包括九个成员:IRF-1、IRF-2、IRF-9/ISGF3γ、IRF-3、IRF-4 (Pip/LSIRF/ICSAT)、IRF-5、IRF-6、IRF-7 和 IRF-8/ICSBP。全部 IRF 蛋白均在其氨基末端 DNA 结合结构域共有同源性。IRF 家族成员通过与共有相似 DNA 结合基序(如 IFN 刺激反应元件 (ISRE)、IFN 共有序列 (ICS) 和 IFN 调节元件 (IRF-E))的蛋白质相互作用而调节转录 (2)。
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