Cat. # | Size | Price | Inventory |
---|---|---|---|
5528T | 20 µl | ||
5528S | 100 µl |
REACTIVITY | H M R Mk B Pg |
SENSITIVITY | Endogenous |
MW (kDa) | 90 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Simple Western™ | 1:10 - 1:50 |
Immunohistochemistry (Paraffin) | 1:800 - 1:3200 |
Immunofluorescence (Immunocytochemistry) | 1:200 - 1:400 |
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: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
RECOMMENDED DETECTION REAGENTS |
SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) #8114 | SignalStain® Boost IHC Detection Reagent (AP, Rabbit) #18653 |
---|---|---|
COMPATIBLE CHROMOGEN |
SignalStain® DAB Substrate Kit #8059 | SignalStain® Vibrant Red Alkaline Phosphatase Substrate Kit #76713 |
SignalStain® Vivid Purple Peroxidase Substrate Kit #96632 | SignalStain® Ultra Blue Alkaline Phosphatase Substrate Kit #12824 | |
SignalStain® Deep Black Peroxidase Substrate Kit #72986 | ||
SignalStain® Radiant Yellow Peroxidase Substrate Kit #69644 |
NOTE: Use of detection reagents other than those specified in this protocol may require further optimization of the primary antibody to account for the different sensitivities of the detection reagents.
posted February 2010
revised June 2020
Protocol Id: 283
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, 牛 , 猪
犬 , 马, 兔
使用与人 RSK2 蛋白中 Pro686 周围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。
90 kDa 核糖体 S6 激酶 (RSK1-4) 是一个广泛表达的丝氨酸/苏氨酸激酶家族,其特征是包含 2 个不同的功能性激酶结构域 (1) 和一个胞外信号调节激酶 (ERK) 羧基末端锚定位点 (2)。RSK 激酶结构域内外的许多位点(包括 Ser380、Thr359、Ser363 和 Thr573)对激酶激活非常重要 (3)。在许多生长因子、多肽激素和神经递质的刺激下,MAPKs 磷酸化、自磷酸化以及磷酸肌醇-3-OH 激酶 (PI3K) 会协同激活 RSK1-3 (3)。
不同生长因子的刺激会激活 RSK2,这是多个通路中的一个关键下游效应子激酶。EGF 刺激会导致 CREB在Ser133 位点以及组蛋白 H3 在体内被 RSK2 磷酸化 (4,5)。RSK2 磷酸化 p53 有助于调节染色质结构和细胞周期 (6)。RSK2 主要在脑细胞中表达,并且对认知功能和学习非常重要。在发育期间,RSK2 可调节成骨细胞和骨骼肌细胞的分化 (7,8)。相应基因突变与 Coffin-Lowry 综合征 (CLS) 有关,这是一种 X 连锁疾病,表现为智力迟钝和面部特征异常 (9)。
探索与本品相关的通路。
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