提升蛋白免疫印迹Western Blot灵敏度10

作者：张馨予

提升蛋白免疫印迹Western Blot灵敏度10-1000倍，缩短至4.5小时

2022-08-19 14:28 来源: 乐备实LabEx

原标题：提升蛋白免疫印迹Western Blot灵敏度10-1000倍，缩短至4.5小时

MSD ( Meso Scale Discovery)电化学发光免疫分析技术的多阵列(Multi-Array)技术为生命科学、临床前和临床研究的生物标志物测量免疫分析的开发提供了一个极好的平台（超敏多因子电化学发光分析仪：SQ120 & S600）。MSD分析具有超低检测限，提供最多5个数量级的线性动态范围，使用最小样本，并轻松处理不同类型复杂样品。该技术的核心是专门开发的微孔板，以充分利用该平台的独特优势。本系列描述了如何将不同类型的抗原（细胞裂解液，细胞膜，悬浮细胞，碳水化合物等）固定在MSD独家专利的石墨96孔板微孔板内，利用MSD电化学发光免疫分析技术优势，开发新的单分析物分析方法。

药物研究及基础科研人员经常使用Western Blot（WB），但WB需要跑胶，转膜，封闭，一抗，二抗，压片等步骤，步骤非常繁琐。流程用最先进的方法也要整整一天，而且还要考虑各个步骤的效率来确认实验成功。一些高丰度的蛋白，超过WB检测上限，信号过强，需要重新摸索实验条件。一些低丰度的蛋白，又低于WB检测下限，检测不到。在药物筛选及药物活性检测项目中，需要鉴定一些细胞内的蛋白从而检测IC50，对于重复性要求很高，而WB的重复性又很差。

很多研究者使用Sigma, Abcam, Millipore,CST等品牌抗体，并自行使用三种不同类型的MSD石墨微孔板(high bind, Standard Bind , Streptavidin)开发检测方案，成功获得了比WB更理想的数据，达到研究目的。

案例一：MSD高载量石墨板 + WB抗体 + MSD Sulfotag标记二抗

文章：Baader, Manuel, et al. "Characterization of the properties of a selective, orally bioavailable autotaxin inhibitor in preclinical models of advanced stages of liver fibrosis." British journal of pharmacology 175.4 (2018): 693-707.

样品处理：Liver extracts (50 mg) were prepared by suspending in 700 μL of MSD lysis buffer (Meso Scale Discovery, USA) supplemented with protease inhibitor cocktails (Thermo Fisher Scientific and Sigma-Aldrich). Samples were homogenised for 30 sec at 6000 g, 4°C, using a FastPrep homogeniser (MP Biomedicals, USA). Homogenates were centrifuged at 10000 g at 4°C for 10 min. Supernatants were adjusted to a protein concentration of 3 mg × mL-1.

检测指标：An MSD western-replacement method (Meso Scale Discovery) using specific anti-alpha smooth muscle actin (alpha-SMA) antibody was used to quantify SMA in protein lysates. Samples (25 uL) were added to multi-array 96-well plates (high bind, Meso Scale Discovery) and incubated for 2 h at room temperature with gentle shaking. Non-specific antibody binding was prevented by incubation of 150 μL of 3% blocking buffer (0.6 g of MSD Blocker A in 20 mL bidest) for 1 h at room temperature. For the detection of bound alpha-SMA, an anti-alphaSMA antibody (1:5000; Sigma-Aldrich) and goat anti-mouse sulfo-TAG antibody (1:167; Meso Scale Discovery) mixture was prepared in blocking buffer (1.5 mL, 3%) and Tris (4.47 mL, pH 7.4) wash buffer and 25 μL added per well for 1 h at room temperature. Plates were washed three times using 200 μL of Tris pH 7.4 wash buffer between all steps. Final detection reaction was initiated by the addition of 150 μL of MSD read buffer T after which plates were analysed on a SECTOR S 600 plate reader (Meso Scale Discovery)

案例二：MSD标准石墨板 + WB抗体 + MSD Sulfotag标记二抗

提升蛋白免疫印迹Western Blot灵敏度10

文章：Activation of Liver AMPK with PF-06409577 Corrects NAFLD and Lowers Cholesterol in Rodent and Primate Preclinical Mode

样品处理：Liver tissue samples were homogenized in cell lysis buffer (Cell Signaling).

检测指标：For AMPK MSD assays Standard Bind plates (Meso-Scale Diagnostics, L15XA-3) were coated overnight at 4 °C with 120 ng/well of AMPKα antibody (Abcam, 80,039). Each well was washed once with 200 μL 1× MSD Tris Wash Buffer (Meso-Scale Diagnostics, R61TX-1) then 200 μL of 1% Blocker A (Meso-Scale Diagnostics, R3AA-2), diluted in 1× Tris Wash Buffer, was added to each well and incubated at 37 °C for 1 h. Wells were washed and 25 μg of the protein samples of interest were added to each well, then incubated at 37 °C for 2 h. Wells were then washed three times with 200 μL of 1× Tris Wash Buffer, then either phospho AMPK antibody (Cell Signaling, 2535) at 25 ng/well or total AMPK antibody (Cell Signaling, 5831) at 100 ng/well, diluted in 1% MSD Blocker A, were added to respective wells and incubated for 1.5 h at 37 °C. Wells were then washed with 1× Tris Wash Buffer, once, then 50 ng of MSD Sulfo-Tag antibody (Meso-Scale Diagnostics, R32AB-1) was added to each well and incubated at 37 °C for 1 h. Wells were washed three times with 1×Tris Wash Buffer, 150 μL of 1× MSD Read Buffer (Meso-Scale Diagnostics, R92TC-2) was added to each well and plate read on an MSD plate reader (Meso-Scale Diagnostics, Sector S600).For ACC MSD assays, MSD Streptavidin gold plates (Meso-Scale Devices, L15SA-2) were blocked in 1% Blocker A (Meso-Scale Diagnostics, R3AA-2), diluted in 1× Tris Wash Buffer, and incubated at 37 °C for 1 h. Following incubation, wells were washed three times with 200 μL of 1× Tris Wash Buffer, 25 μg of the protein samples of interest were added to each well, then incubated at 37 °C for 2 h. Wells were then washed three times with 200 μL of 1× Tris Wash Buffer, then either phospho ACC antibody (Millipore, 07–303) at 50 ng/well or total ACC antibody (Cell Signaling, 3676) at 25 ng/well, diluted in 1% Blocker A, were added to respective wells and incubated for 1.5 h at 37 °C. Wells were then washed with 1× Tris Wash Buffer, once, then 50 ng of MSD Sulfo-Tag antibody (Meso-Scale Diagnostics, R32AB-1) was added to each well and incubated at 37 °C for 1 h. Wells were washed three times with 1× Tris Wash Buffer, 150 uL of 1× MSD Read Buffer (Meso-Scale Diagnostics, R92TC-2) was added to each well and plate read on an MSD plate reader (Meso-Scale Diagnostics, Sector S600).

和普通WB相比，MSD 电化学发光检测的优势: