Cat. # | Size | Price | Inventory |
---|---|---|---|
33402S | 100 µl |
REACTIVITY | M |
SENSITIVITY | Endogenous |
MW (kDa) | 42-62 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:100 |
Flow Cytometry (Fixed/Permeabilized) | 1:400 - 1:1600 |
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
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity utilizing Protein A magnetic separation.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.
NOTE: Add 1 mM PMSF (#8553) immediately prior to use.
A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein A Magnetic beads. Pre-clear enough lysate for test samples and isotype controls.
IMPORTANT: Pre-wash #73778 magnetic beads just prior to use:
Carefully remove the buffer once the solution is clear. Add 500 μl of 1X cell lysis buffer to the magnetic bead pellet, briefly vortex to wash the beads. Place tube back in magnetic separation rack. Remove buffer once solution is clear. Repeat washing step once more.
IMPORTANT: The optimal lysate concentration will depend on the expression level of the protein of interest. A starting concentration between 250 μg/ml-1.0 mg/ml is recommended.
IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 for rabbit monoclonal primary antibodies, Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal IgG1 primary antibodies, Mouse (E5Y6Q) mAb IgG2a Isotype Control #61656 for mouse monoclonal IgG2a primary antibodies, Mouse (E7Q5L) mAb IgG2b Isotype Control #53484 for mouse monoclonal IgG2b primary antibodies, and Mouse (E1D5H) mAb IgG3 Isotype Control #37988 for mouse monoclonal IgG3 primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples.
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2008
revised April 2021
Protocol Id: 410
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
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro266 of mouse ZBP1 protein.
Z-DNA 结合蛋白 1 (ZBP1) 也称为 DNA 依赖性 IFN 调节因子激活蛋白 (DAI) 和 DLM-1,它是一种在肿瘤发生和对病毒感染作出天然免疫应答方面起作用的核苷酸结合蛋白 (1)。它在淋巴组织和肠内表达水平高,并且干扰素 γ 或 LPS 会诱导巨噬细胞表达 (2,3)。It contains two amino-terminal Z-alpha domains that bind to left-handed Z-DNA and Z-RNA (4,5). 此外,一个邻近的结构域还会结合右侧 B-DNA,B-DNA 可让其在天然免疫应答中作为胞质感应蛋白起作用,从而触发 TBK1 和 IRF-3 的激活,以及随后产生 I 型干扰素 (6,7)。此外,ZBP1 还会与 RIPK1 和 RIPK3 的 RIP 同型互作基序 (RHIM) 相互作用,从而触发 NF-κB 的激活 (8)。ZBP1 结合 RIPK3 还会诱导一个称为坏死性凋亡的程序性坏死过程 (9)。相比之下,其与 RIPK1 的相互作用可抑制坏死性凋亡 (10,11)。ZBP1 结合mRNA 的活性还被认为在肿瘤发生中起作用。ZBP1 在转移性乳腺癌细胞中受到抑制,导致与增殖和转移有关的 mRNA 靶标失调 (12,13)。
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