斑点印迹（Dot blot）
By Kaiyi
实验原理
在斑点印迹中，要检测到的生物分子（DNA、RNA和蛋白均可）无需通过凝胶电泳分离，而是将含有被检测分子的混合物直接点在膜上，晾干固定；再用核酸探针或抗体杂交后，即可检测。这种方法耗时短，可半定量。一张膜上可同时检测多个样品，实现高通量。
实验材料与仪器
1. 材料
ac4c抗体（Abcam,1:1000），二抗IgG，带有正电荷的尼龙膜（索莱宝），TBST，5%BSA或者脱脂牛奶，0.1%亚甲蓝（索莱宝），显影液。
2. 仪器
金属浴，紫外交联仪（可使用紫外灯管替代），曝光仪。
参考文献（RNA ac4c dot blot）
ac4C detection by dot blot
参考文献1：Acetylation of Cytidine in mRNA Promotes Translation Efficiency
Dot blots were performed using rabbit monoclonal anti-ac4C antibodies as described previously (Sinclair et al., 2017). Briefly, 1-10 mg RNA were denatured at 75℃ for 5 min, immediately placed on ice for 1 min and loaded onto Hybond-N+ membranes. Membranes were crosslinked twice with 150 mJ/cm2 in the UV254nm Stratalinker 2400 (Stratagene), blocked with 5% non-fat milk in 0.1% Tween20 PBS (PBST) for 30 min at room temperature, and probed overnight with anti-ac4C antibody in 1% non-fat milk (1:1000) at 4℃. Membranes were next washed three times with 0.1% PBST, incubated with HRP-conjugated secondary anti-rabbit IgG in 1% nonfat milk (1:10000 dilution, Cell Signaling Technology) at 4℃ overnight, washed four times with 0.1% PBST and developed with the SuperSignal ELISA Femto Maximum Sensitivity Substrate (ThermoScientific).
参考文献2：NAT10 promotes vascular remodelling via mRNA ac4C acetylation
Briefly, 5-10 μg total RNA extracted from tissue or cell samples were denatured at 95 °C for 3 minutes to disrupt secondary structures and then immediately chilled on ice. Then, 2 μL of total RNA were directly dropped onto the Hybond-N+ membrane (GE Healthcare, RPN203B), optimized for nucleic acid transfer. The RNA was crosslinked to the membrane in a Stratalinker (Scientz Co., Ltd) 2400 UV Crosslinker twice, with each crosslinking step consisting of 1,200 microjoules [×100] for 25-50 seconds. The membrane was then blocked with 5% non-fat milk in 0.1% Tween-20 PBS (PBST) for 1 hour at room temperature. Subsequently, it was incubated with anti-ac4C antibody (Abcam, ab252215, 1:1000) in 5% non-fat milk at 4 °C overnight. After three washes with 0.1% PBST, the membrane was incubated with HRP-conjugated secondary anti-rabbit IgG (Abclonal, AS014) in 5% non-fat milk at room temperature for 1 hour. Following three additional washes with 0.1% PBST, the target dot on the blots was visualized using the Ultra-High Sensitivity ECL Kit, and images were acquired using the ChemiDoc Imaging Systems. For internal standard detection, the membranes were incubated with a staining solution containing 0.2% methylene blue (MB, Sigma-Aldrich, M4159) in 0.4 M sodium acetate. The band densities were quantified using the Image J software (NIH).
实验步骤
1、调齐浓度
首次实验可以在浓度允许的情况下使用多个浓度，推荐稀释浓度为400ng/ul，200ng/ul和100ng/ul，最低浓度最好不要低于100ng/μL。
因后续点样时推荐点1μL或者2μL，稀释样品时最好稀释出5μL，以防后续过程中的消耗（低浓度的RNA更容易降解，因此最好短时间内使用完稀释的样品或者现用现配）。
2、变性
样品稀释好后放入95℃金属浴3分钟，使mRNA变性以破坏其二级结构。
结束后使用掌上离心机瞬离管壁上的液体。
立刻置于冰上1分钟，防止二级结构形成。
3、 上样
裁剪尼龙膜（Hybond-N+ membrane），用铅笔在膜上轻轻标记网格，以指示样本加载位置。将膜转移到干净的直径为10cm皿中。
滴加2μL样品到尼龙膜上。点样时尤其是多个样品时加快速度，缩短点样到交联的时间。
滴加样品时注意让RNA自然扩散，一定避免枪头戳到尼龙膜，整个实验过程中同时也要避免镊子或者其他尖锐物品接触点样区域。
4、交联
立即放置样品于紫外交联仪中或者紫外灯下，使RNA面朝上，交联30min。
若使用紫外灯管替代紫外交联仪时可以使用枪头盒或其他物品将尼龙膜垫高。
5、封闭
用10 mL TBST（1X TBS，0.1% Tween-20）洗膜，室温下轻轻摇动5分钟，洗去未结合的RNA。
放入5%脱脂牛奶0.1% Tween20的TBST中封闭1h。
6、敷一抗
用含5%脱脂奶粉0.1% Tween20的TBST配置一抗。
RNA面朝下孵育ac4c一抗，置于摇床（转速50左右）上4℃过夜。
7、洗涤
回收一抗，翻转膜，使RNA面朝上。
TBST置于摇床上轻柔（50-100转）清洗3遍，每次5min。
8、敷二抗
RNA面朝下孵育二抗，常温摇床（转速50左右）孵育1h。
清洗二抗，TBST置于摇床上轻柔（不要超过100转）清洗3遍，每次5min。
9、显影
配制显影液，A液B液等量混合现用现配。
滴加显影液至点样的大概位置，将显影液铺匀，尼龙膜上下都不应有气泡，曝光。
10、亚甲基蓝染色
提前用0.4mol/L的乙酸钠溶液配置0.2%的亚甲基蓝溶液。
将尼龙膜放入亚甲基蓝溶液（0.1%成品溶液稀释5倍后使用）中染色10分钟，置于摇床上轻柔摇晃（转速50左右）。
使用单蒸水清洗尼龙膜，明场拍照，作为内参。
曝光结束后推荐将尼龙膜放入1XSSC溶液中浸泡一段时间，会有更好的亚甲蓝染色效果。
亚甲基蓝很难清洗，实验操作中要记得戴口罩和穿白大褂。在进行亚甲基蓝染色时，注意尽量不要吸取底部沉淀。
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