westernblot详细实验步骤（如何恰当地描述Western）

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专家意见：western blot section is lacking and requires more details.

译文： Western blot方法部分缺乏足够的信息。

编辑解读：

Western blot流程如下：

作者在向NRR投稿时，需要注意Western blot方法的描述，其基本要求如下：

从XX细胞/XX组织中提取蛋白质，用RIPA裂解和提取缓冲液（KeyGen Biotechnology，中国南京）处理。用BCA方法测定蛋白浓度。等量蛋白质通过SDS-PAGE分离后，转移到PVDF膜上（EMD Millipore公司，Burlington, MA），然后用牛血清白蛋白（BSA，5%，v/v）封闭膜 。用适当稀释度的一抗在4℃下过夜孵育膜，然后将膜与二抗在室温下孵育1小时。ECL化学发光试剂盒（Thermo Fisher Scientific）进行曝光显影。最后使用ImageJ（National Institutes of Health, Bethesda, MD, USA）对蛋白条带进行半定量分析，最终结果以目的蛋白与内参的吸光度比值表示（蛋白归一）。(注：一抗和二抗应说明抗体名称，动物种属，生产厂家（城市、国家），工作浓度，目录号及RRID号)

下面是Western blot方法描述较好的样例：

Western blot assay

IL-6, IL-1β and TNF-α expression levels were measured to evaluate the inflammatory response in spinal cord tissue and cells. NLRP3, apoptosis-assocIAted speck-like protein containing a CARD (ASC), pro-Caspase-1, Caspase-1, GSDMD, and IL-18 levels were measured to evaluate pyroptosis. Nrf2 and HO-1 levels were measured to assess the Nrf2/ HO-1 signaling axis. First, 100 mg of spinal cord tissue was washed with PBS, fully lysed with radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China) containing phenylmethylsulfonyl fluoride for 30 minutes, incubated on ice, and immediately centrifuged at 4°C. The supernatants were then collected, and the protein concentrations was determined using the bicinchoninic acid method (Abdulrahman et al., 2018) with the PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific). The samples were loaded onto a polyacrylamide gel and electrophoresed. Next, the proteins were transferred to polyvinylidene fluoride membranes and blocked in 5% non-fat milk for 2 hours. Subsequently, the membranes were incubated overnight at 4°C with rat monoclonal primary antibodies against IL-6 (1:1000, Acris Antibodies GmbH, Herford, Germany; Cat# SM1695PX, RRID:AB_1004115), IL-1β (1:100, Miltenyi Biotec, Bergisch Gladbach, Germany; Cat# 130-125-220, RRID:AB_2889697), T N F - α ( 1 : 1 0 0 , M i l te ny i B i o te c ; C at # 1 3 0 - 1 0 2 - 3 8 6 , RRID:AB_2661141), NLRP3 (1:1000, MyBioSource, San Diego, CA, USA; Cat# MBS604215, RRID:AB_10911109), ASC (1:2000, Gabriel Núñez, University of Michigan, Ann Arbor, MI, USA; Cat# GN-ASC, RRID:AB_2750645), pro-Caspase-1 (1:1000, ProSci, San Diego, CA, USA; Cat# 48-495, RRID:AB_1945485), C a s p a s e - 1 ( 1 : 1 0 0 0 , A c r i s A n t i b o d i e s G m b H ; C a t # AP06610PU-N, RRID:AB_1611115), GSDMD (1:100, Thermo Fisher Scientific; Cat# A305-736A-M, RRID:AB_2782894), and IL-18 (1:100, DSHB, Iowa City, IA, USA; Cat# CPTC-IL18-2, RRID:AB_1553716), followed by incubation with horseradish peroxidase–conjugated rabbit anti-rat IgG (1:1000, Biorbyt, Cat# orb21564, RRID:AB_10932535) at 20°C for 1 hour, and finally stained with a diaminobenzidine solution. A Bio-Image (Bio-Rad Laboratories, Hercules, CA, USA) gel imaging system was used to collect images. The optical density was measured using ImageJ software. Glyceraldehyde-3-phosphate dehydrogenase (1:1000, LSBio (LifeSpan), Seattle, WA, USA; Cat# LS-C94067-150, RRID:AB_1932766) and β-actin (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA; Cat# sc-47778 HRP, RRID:AB_2714189) were used as internal references.

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专家意见： The entire gel must be provided as supplementary results。

译文：提供原始western blot凝胶图像作为补充资料。

编辑解读：作者在提交稿件时，需要提供原始的、未经裁剪和最小化调整的印迹和凝胶图像（提供原始数据或第三方链接）作为数据真实性的证据。

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专家意见： The images for the representative blots in figure 3A were inappropriately contrast-adjusted. No-background blots of this sort are not allowed.

译文：图3A中代表印迹的图像被不适当地调整了对比度。这类无背景印迹是不允许的。

编辑解读：在对western blot凝胶图像进行加工处理时，需要注意不得以任何方式调整图像，以免影响显示的科学信息。背景和对比度不应该加以修改以明显改变数据可见度、背景或非特异性带。

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专家意见：This should be 0.2 μm pore size for western blots rather than 0.2 mm.

译文：这应该是0.2μm孔径，而不是0.2mm。

编辑解读：审稿专家提到的孔径是针对PVDF膜的，注意在描述时的严谨性和准确性。