Comprehensive Optimization of Western Blotting
蛋白质印迹技术的综合优化

Abstract 摘要

Western blotting is one of the most extensively used techniques in the biomedical field. However, it is criticized by many researchers due to its considerable time consumption, multiple steps, and low method results. Therefore, we modified the steps of gel preparation, electrophoresis, electrotransfer, blocking, and gel cutting. First, we simplified the gel preparation step by premixing various reagents and varying the amounts of catalysts or radical generators, which shortened the entire process to 10 min. Second, we shortened the electrophoresis process to 35 min by modifying the formula of the electrophoresis running buffer. Then, we removed the hazard of methanol vapor by replacing methanol with ethanol in the electrotransfer buffer. Finally, the use of polyvinylpyrrolidone-40 shortened the blocking procedure to 10 min. Our modifications shortened the time, improved the experimental productivity, and minimized the experimental cost without hindering compatibility with most existing equipment. The entire experiment up to primary antibody incubation can be completed within 80 min.
蛋白质印迹技术是生物医学领域应用最广泛的技术之一。然而，由于其耗时大、步骤多、方法结果低，受到了许多研究人员的批评。为此，我们对凝胶制备、电泳、电转移、封闭、凝胶切割等步骤进行了改进。首先，我们简化了凝胶的制备步骤，通过预混各种试剂和改变催化剂或自由基生成剂的量，将整个过程缩短到10分钟。其次，通过修改运行缓冲液的配方，将电泳法的运行时间缩短到35min。然后，我们通过在电转移缓冲液中用乙醇取代甲醇来消除甲醇蒸气的危害。最后，使用聚乙烯吡咯烷酮-40可将阻断过程缩短至10分钟。我们的改进缩短了时间，提高了实验生产率，并在不妨碍与大多数现有设备兼容的情况下将实验成本降至最低。整个实验直至一次抗体孵育均可在80分钟内完成。

1. Introduction 1.介绍

Western blotting (WB) is an essential tool in protein analytical chemistry [1,2]. However, its shortcomings, such as considerable time consumption, multiple steps, and poor reliability, are criticized by researchers [3,4]. Even small mistakes in any of its steps may significantly alter the entire subsequent experiment. Therefore, researchers who invested considerable time and effort in mastering this approach do not necessarily obtain satisfactory results.
蛋白质印迹(WB)是蛋白质分析化学的重要工具[1，2]。然而，其耗时大、步骤多、可靠性差等缺点受到了研究人员的批评[3，4]。它的任何一个步骤中的小错误都可能显著改变随后的整个实验。因此，投入大量时间和精力掌握这一方法的研究人员并不一定能获得满意的结果。

The principle of immunoblotting is not particularly complicated. First, mixed antigen samples are separated by unidirectional or bidirectional electrophoresis on a gel. Then, the single antigenic component in the gel is transferred to the blotting membrane and solidified by natural adsorption, an electric field, or other external forces of the blotting membrane. Finally, the antigen-immobilized matrix membrane is detected and analyzed by using immunoisotope probes or immunoenzymatic probes [5]. However, these simple principles require various steps.
免疫印迹的原理并不是特别复杂。首先，混合抗原样品在凝胶上通过单向或双向电泳法分离。然后，凝胶中的单一抗原组分转移到印迹膜上，并通过印迹膜的自然吸附、电场或其他外力固化。最后，用免疫同位素探针或免疫酶探针对抗原固定的基质膜进行检测分析[5]。然而，这些简单的原则需要不同的步骤。

Immunoblotting includes core steps, such as gel preparation, electrophoresis, electrotransfer, blocking, and antibody incubation. Each step lasts hours or days. Despite the fact that commercial tricine-based precast gels have been developed [6,7], a high price is not appropriate for high-output experiments. Therefore, primitive methods, such as adding reagents one by one and slow solidification, are still used to formulate gels in most laboratories. In addition, traditional electrophoresis (stacking gel: 80 V; separation gel: 120 V) takes at least 90 min, and wet electrotransfer also requires 90 min. At least 60 min are required for skim milk blocking before primary antibody incubation [8]. This five-hour-long experimental process truly needs to be optimized.
免疫印迹包括核心步骤，如凝胶制备、电泳、电转移、封闭和抗体孵育。每一步都要持续几小时或几天。尽管已经开发了基于三氯甲烷的商业预制凝胶[6，7]，但对于高产量的实验来说，高昂的价格并不合适。因此，大多数实验室仍然使用逐个添加试剂、缓慢固化等原始方法来配制凝胶。此外，传统的电泳法(堆积胶：80V；分离胶：120V)至少需要90分钟，湿法电转移也需要90分钟。在一次抗体孵育前，至少需要60分钟来阻断脱脂奶[8]。这个长达五个小时的实验过程确实需要优化。

Although all-in-one instruments, such as ProteinSimple Wes, are gradually gaining ground in the market [9,10,11], their high price is prohibitive for many laboratories. Other semi-automatic systems, such as SNAP i.d.* 2.0 [12] and Invitrogen™ iBind™, have been developed to accelerate the protein detection process at the expense of consuming large quantities of precious antibodies.
尽管ProteinSimple Wes等一体化仪器在市场上逐渐占据一席之地[ 9，10，11]，但其高昂的价格让许多实验室望而却步。其他半自动系统，例如SNAP i. d. * 2.0[ 12]和Invitrogen™ iBind™，旨在加速蛋白质检测过程，但代价是消耗大量珍贵抗体。

No matter how often these instruments are upgraded, the fundamentals of immunoblotting remain the same. In this work, we modified the gel preparation, electrophoresis, membrane transfer, blocking, and gel cutting steps. While being compatible with most existing instruments, our modifications shorten the time needed, improve the experimental success rate and productivity, and also decrease the experimental cost. The entire experiment can be completed within 80 min before the primary antibody incubation, which dramatically improves the labor and productivity of the experimenter.
无论这些仪器升级得多频繁，免疫印记的基本原理都保持不变。在这项工作中，我们修改了凝胶制备、凝胶、膜转移、封闭和凝胶切割步骤。在与大多数现有仪器兼容的同时，我们的修改缩短了所需时间，提高了实验成功率和生产力，并降低了实验成本。整个实验可以在一抗温育前80分钟内完成，极大地提高了实验人员的劳动力和生产力。

2. Results 2.结果

2.1. Pre-Mixed Reagents Simplify and Accelerate Gel Preparation
2.1.预混试剂可简化和加速凝胶的制备

Considering the role of each component in gel preparation, the compounding scheme can be simplified. We used 10% separating gel (1.5 M Tris (pH 8.8), 1.0 mm) and 5% stacking gel ((1.0 M Tris (pH 6.8), 1.0 mm) as an example. The reagents required for gel preparation were deionized water, 30% Acr-Bis, Tris, 10% SDS, TEMED, and 10% AP. TEMED catalyzes the generation of free radicals from AP, which initiates the crosslinks between bisacrylamide and acrylamide, forming a three-dimensional network. Hence, the isolation of free radicals enables the stable preservation of the mixed solution. Our results show that the first five reagents can be mixed into a system and stored at 4 °C in the dark for a month or more. In this way, the entire compounding steps are simplified to only adding the mixed system and AP. Similarly, dye indicators, which are usually added to form a pre-stained gel for easy sample loading, can also be added to the mixed system without reaction [13]. In order to make the gel agglomeration faster, AP or TEMED could be appropriately adjusted to catalyze the reaction. We compared the traditional step-by-step-added reagent gel, the pre-mixed reagent gel, and the pre-mixed reagent gel at 4 °C overnight. Our results demonstrate no substantial differences in the signal-to-noise ratio among the three gels (Figure 1A,B).
考虑到各组分在凝胶制备中的作用，可以简化配制方案。以10%分离凝胶(1.5M Tris(pH 8.8)，1.0 mm)和5%堆积凝胶(1.0M Tris(pH 6.8)，1.0 mm)为例。凝胶制备所需试剂为去离子水、30%ACR-Bis、Tris、10%十二烷基硫酸钠、TEMED和10%AP。TEMED催化AP产生自由基，引发双丙烯酰胺和丙烯酰胺之间的交联，形成三维网络。因此，自由基的分离使混合溶液得以稳定保存。我们的结果表明，前五种试剂可以混合到一个系统中，并在4℃的黑暗中保存一个月或更长时间。这样，整个复合步骤简化为只加入混合体系和AP。同样，染料指示剂也可以加入到混合体系中而不发生反应[13]，这些指示剂通常被添加到预染色凝胶中，以便于样品加载。为了使凝胶团聚更快，可以适当调整AP或TEMED来催化反应。我们比较了传统的分步加入试剂凝胶、预混合试剂凝胶和4°C下的预混合试剂凝胶。我们的结果表明，三种凝胶之间的信噪比没有显著差异(图1A，B)。

Figure 1. Pre-mixed gels and modified electrophoresis running buffer accelerate the experimental procedures. (A) Comparison of signal intensities after the development of traditional gels, quick-formulated gels, and quick-formulated gels at 4 °C overnight. The experimental procedure was the same as the standard experimental procedure except for the gels. (B) Signal intensities after the development of different gel preparation schemes were statistically analyzed and compared (n = 6). There was no substantial difference in signal-to-noise ratio between the gels prepared using the different methods. (C) The traditional electrophoresis running buffer tested at 200 V. The experimental procedure was the same as the standard experimental procedure except for the electrophoresis running buffer. (D) Modified electrophoresis running buffer tested at 200 V. The experimental procedure was the same as the standard experimental procedure except for the blocking reagent (Epizyme, Shanghai, China). (E) Signal intensities of different voltages in the traditional electrophoresis running buffer were statistically analyzed and compared (n = 6). Traditional electrophoresis running buffer resulted in a loss of protein signals as the voltage increased. (F) Signal intensities of commercial blocking reagents were statistically analyzed and compared (n = 4). The standard experimental procedure was compatible with commercially blocking reagent, and the protein signal intensity showed a linear relationship with the sample loading mass within a certain scope. (G) Modified electrophoresis running buffer tested under different voltage conditions. The experimental procedure was the same as the standard experimental procedure except for the voltage of the modified electrophoresis running buffer.
图1.预混合凝胶和改良的电泳运行缓冲液加快了实验过程。(A)传统凝胶、快速配方凝胶和快速配方凝胶在4°C下显影一夜后信号强度的比较。除凝胶剂外，实验程序与标准实验程序相同。(B)统计分析和比较不同凝胶制备方案形成后的信号强度(n=6)。使用不同方法制备的凝胶之间的信噪比没有实质性差异。(C)传统的运行缓冲液在200V下进行测试。除运行缓冲液外，实验程序与标准实验程序相同。(D)在200V下测试的改良电泳运行缓冲液。除封闭剂(上海伊匹西姆，中国)外，实验程序与标准实验程序相同。(E)对传统运行缓冲液中不同电压下的信号强度进行统计分析和比较(n=6)。传统的电泳运行缓冲液随着电压的升高而导致蛋白质信号的丢失。(F)对市售封堵剂的信号强度进行统计分析和比较(n=4)。标准实验程序与市售封闭剂兼容，蛋白质信号强度在一定范围内与样品量呈线性关系。(G)在不同电压条件下测试改良的电泳运行缓冲液。除改良的电泳运行缓冲液的电压外，实验程序与标准实验程序相同。

2.2. Modification of the Formula of the Running Buffer to Accelerate Electrophoresis
2.2.改进运行缓冲液配方以加速凝胶

We tested the traditional electrophoresis running buffer (Tris 19.2 mM, glycine 19.2 mM, SDS 3.5 mM, and pH 8.3) at a high voltage. Regrettably, various problems occurred with the voltage increase. One of the most notable drawbacks is that it lost protein signals and could not significantly shorten the electrophoresis time (Figure 1C,E). In order to accelerate the electrophoresis, we modified the formula of the running buffer (Tris 38.1 mM, glycine 266.7 mM, HEPES 21.0 mM, SDS 3.5 mM, and pH 8.3). The modified running buffer could complete electrophoresis within 35 min under 200 V at room temperature (Figure 1D,F) (Table 1). Subsequently, we tested the modified electrophoresis buffer under different voltages (Table 1). Surprisingly, when the voltage was set at 300 V, it still worked normally (Figure 1G), but the ensuing rising heat required an ice-water bath to dissipate it. The 1× electrophoresis buffer was prone to flocculation after storage at room temperature. Certainly, the 10× electrophoresis buffer does not deteriorate easily, but it is difficult to dissolve due to the large amount of electrolytes. Generally, we prepared a 5× electrophoresis buffer for stockpiling.
我们在高压下测试了传统的电泳运行缓冲液(Tris 19.2 mm，甘氨酸19.2 mm，十二烷基硫酸钠3.5 mm，pH 8.3)。令人遗憾的是，随着电压的增加，出现了各种问题。最显著的缺点之一是它失去了蛋白质信号，并且不能显著缩短电泳时间(图1C，E)。为了加快电泳速度，我们修改了运行缓冲液的配方(Tris38.1 mM，甘氨酸266.7 mM，HEPES21.0 mM，SDS3.5 mM，pH 8.3)。改进的运行缓冲液在室温下200V下可在35min内完成电泳(图1D，F)(表1)。随后，我们测试了不同电压下的修饰后的电泳液(表1)。令人惊讶的是，当电压设置为300V时，它仍然正常工作(图1G)，但随之而来的不断上升的热量需要冰水浴来驱散。1倍电泳缓冲液在室温保存后易发生絮凝。当然，10倍的电泳液不容易变质，但由于电解液的量较大，很难溶解。一般情况下，我们准备了5倍的电泳液用于储藏。

Table 1. Changes in electrophoresis time and pH of the modified formula at different voltages.

Voltage	Time (RT/LT min)	RT pH (Internal Buffer/External Buffer)	LT pH (Internal Buffer/External Buffer)
150 V	45/55	8.52/8.31	8.47/8.31
200 V	35/40	8.59/8.30	8.48/8.33
250 V	23/29	8.53/8.39	8.59/8.31
300 V	22/27	8.64/8.32	8.57/8.29

Abbreviations: RT, room temperature; LT, low temperature. Note: The electrophoresis time and pH depend on the room temperature. pH was measured by using Sartorius PB-10 (n = 3).

2.3. Replacement of Methanol with Ethanol in the Electrotransfer Buffer to Reduce Toxicity
2.3.用乙醇替代电转移缓冲液中的甲醇以降低毒性

We replaced the methanol with ethanol in the semi-dry electrotransfer buffer (Tris 36.9 mM, glycine 39.1 mM, SDS 1.3 mM, and 20% ethanol). This upgraded formula decreased methanol vapor. Among the tested proteins, the signal-to-noise ratios of GAPDH, CD81, and CyC were not statistically different between the two groups. However, the signal-to-noise ratio of PINK was the strongest in the ethanol group (Figure 2A,B). In addition, we also tested the effect of SDS on electrotransfer efficiency. Our results imply that the addition of SDS enhanced the signal intensities of GAPDH and CD81 (Figure 2A,B).
我们在半干电转移缓冲液（Tris 36.9毫米、Glyine 39.1毫米、SDS1.3毫米和20%乙醇）中用乙醇替代了甲醇。这种升级后的配方减少了甲醇蒸气。在测试的蛋白质中，两组之间GADH、CD 81和CyC的信噪比没有统计学差异。然而，PINK的信噪比在乙醇组中最强（图2A、B）。此外，我们还测试了十二烷基硫酸钠对电转移效率的影响。我们的结果表明，添加SDs增强了GAACH和CD 81的信号强度（图2A、B）。

Figure 2. Effect of electrotransfer buffer formula and different electrotransfer times on different molecular weight proteins. (A) Replacing methanol with ethanol in the electrotransfer buffer achieved the same efficiency. The experimental procedure was the same as the standard experimental procedure except for the electrotransfer buffer. (B) Signal intensities of different electrotransfer buffers were statistically analyzed and compared (n = 6). (C) The effect of different electrotransfer times on the signal intensities of proteins with different molecular weights. The experimental procedure was the same as the standard experimental procedure except for the electrotransfer time. (D) Signal intensities of different electrotransfer times were statistically analyzed and compared (n = 6).
图2.电转移缓冲液配方和不同电转移时间对不同分子量蛋白质的影响。(A)用乙醇代替电转移缓冲液中的甲醇可以获得相同的效率。除了电转移缓冲液外，实验程序与标准实验程序相同。(B)对不同电转移缓冲液的信号强度进行统计分析和比较（n = 6）。(C)不同电转移时间对不同分子量蛋白质信号强度的影响。除了电转移时间外，实验程序与标准实验程序相同。(D)对不同电转移时间的信号强度进行统计分析和比较（n = 6）。

2.4. Proteins of Different Molecular Weights Have the Most Suitable Electrotransfer Time
2.4.不同分子量的蛋白质具有最合适的电转移时间

For proteins with a sufficiently high abundance, the appropriate extension of the electrotransfer time cannot have a significant effect on the signal intensity. As shown in Figure 2C,D, there was no significant statistical difference in the signal intensity of GAPDH at 15 min, 25 min, and 35 min. However, the signal intensity increased with the extension of electrotransfer time for PINK (70 kDa). In contrast, the signal intensity of CyC (15 kDa) decreased with the extension of the electrotransfer time. When the electrotransfer time was extended to 35 min, the protein marker of CyC could not be visualized (Figure 2C). These results show that proteins with low abundance have relatively suitable electrotransfer times. We extensively tested proteins with 15–130 kDa. Based on our experimental experience, we recommend the setting of the following parameters: 10–25 kDa, 25 V, 15 min; 25–55 kDa, 25 V, 20 min; 55–70 kDa, 25 V, 25 min; and 70–130 kDa, 25 V, 30–35 min.
对于丰度足够高的蛋白质，适当延长电转移时间不会对信号强度产生显着影响。如图2C、D所示，GADH在15分钟、25分钟和35分钟的信号强度没有显着的统计学差异。然而，PINK（70 Da）的信号强度随着电转移时间的延长而增加。相比之下，CyC（15 KDa）的信号强度随着电转移时间的延长而减弱。当电转移时间延长至35分钟时，CyC的蛋白标记物无法可视化（图2C）。这些结果表明，低丰度的蛋白质具有相对合适的电转移时间。我们广泛测试了15-130 KDa的蛋白质。根据我们的实验经验，我们建议设置以下参数：10-25 KDa，25 V，15分钟; 25-55 KDa，25 V，20分钟; 55-70 KDa，25 V，25分钟;和70-130 KDa，25 V，30-35分钟。

2.5. The 0.45 μm NC Membrane Intercepts Protein Marker Dyes Better Than the 0.45 μm PVDF Membrane
2.5. 0.45 μm NC膜对蛋白标记物染料的拦截效果优于0.45 μm PDC膜

We conducted a comparative study on a 0.22 μm PVDF membrane, 0.45 μm PVDF membrane, and 0.45 μm NC membrane. Our results show that the 0.45 μm PVDF could not effectively intercept protein marker dye (Figure 3A), and small-molecular-weight proteins were easily over-transferred when large-molecular-weight proteins were transferred into membranes. The advantage is that the background signal is relatively low. The 0.45 μm NC membrane could intercept protein marker dye (Figure 3A), but it had the same over-transferability issue for small-molecular-weight proteins as the 0.45 μm PVDF membrane. The 0.22 μm PVDF membrane not only retained the pre-stained protein marker but also retained proteins of small and medium molecular weights (Figure 3A–C). Through the quantitative analysis of the signal intensity, our results show that the 0.22 μm PVDF membrane had a significantly stronger interception ability for small-molecular-weight proteins (CyC, CD81) than the 0.45 μm PVDF membrane and 0.45 μm NC membrane (Figure 3B,C).
我们对0.22μm聚偏氟乙烯膜、0.45μm聚偏氟乙烯膜和0.45μm NC膜进行了比较研究。结果表明，0.45μm PVDF不能有效地截留蛋白质标记染料(图3A)，当大分子蛋白质转移到膜中时，小分子蛋白质容易过度转移。优点是背景信号相对较低。0.45μm纳米碳化膜能够截留蛋白质标记染料(图3A)，但与0.45μm聚偏氟乙烯膜一样，它对小分子蛋白质也存在过度转移性问题。0.22μm聚偏氟乙烯膜不仅保留了预染色的蛋白质标记，而且还保留了小分子和中等分子量的蛋白质(图3A-C)。通过对信号强度的定量分析，发现0.22μm聚偏氟乙烯膜对小分子蛋白质(CyC、CD81)的截留能力明显强于0.45μm聚偏氟乙烯膜和0.45μm NC膜(图3B，C)。

Figure 3. Effects of different membrane materials and pores on protein signal intensities. (A) The 0.45 μm PVDF cannot effectively intercept protein marker dye. (B) Different membrane materials and pores have different retention capacities for different molecular weight proteins. The experimental procedure was the same as the standard experimental procedure except for the membranes. (C) Signal intensities of different materials of membranes or pores were statistically analyzed and compared (n = 6).
图3.不同膜材料和孔对蛋白质信号强度的影响。(A)0.45μm聚偏氟乙烯不能有效截留蛋白质标记染料。(2)不同的膜材料和孔对不同相对分子质量的蛋白质有不同的截留能力。除膜外，实验程序与标准实验程序相同。(C)对不同材料的膜或孔的信号强度进行统计分析和比较(n=6)。

2.6. Cutting the Gel Also Avoids Band Counterfeiting
2.6.切断凝胶也可以避免带子造假

The immunoblotting band is the hardest-hit area for paper fraud. Every year, myriads of papers are questioned on PubPeer because of protein bands. Gradually, some journals have begun to advocate for uncut gels. Uncut gels undoubtedly increase the workload of researchers and consume reagents and samples. The purpose of an uncut gel is to confirm the target proteins at the corresponding molecular weight and verify the specificity of the primary antibody. With our modified scheme, a cut gel achieved a similar effect. First, we used an oil-based pen to mark the target protein on the membrane after electrotransfer and cut the membrane into bands. Then, the bands were spliced into a whole membrane and an image was acquired. Finally, a brightfield image was preserved during development. As such, photographs plus individual handwriting confirmed the consistency of the bands. As shown in Figure 4, in the bands with falsified trends, the reference protein followed the trend of the target protein. The incompatibility of the reference protein proves that the bands are falsified.
免疫印迹带是纸张欺诈的重灾区。每年都有无数的论文因为蛋白质带而在PubPeer上受到质疑。渐渐地，一些期刊开始倡导使用未切割的凝胶。未切割凝胶无疑增加了研究人员的工作量，消耗了试剂和样本。未切割凝胶的目的是确认相应分子量的目标蛋白，并验证一抗的特异性。在我们的改进方案中，切割凝胶达到了类似的效果。首先，我们用油基笔在电转移后的膜上标记目的蛋白，并将膜切割成条带。然后，将这些条带拼接成一个完整的膜，并获得图像。最后，在开发过程中保存了一张Brightfield图像。因此，照片和个人笔迹证实了这些带子的一致性。如图4所示，在趋势错误的条带中，参考蛋白跟随目标蛋白的趋势。参考蛋白的不亲和性证明这些条带是伪造的。

Figure 4. Cutting the gels avoids band counterfeiting.
图4.切割凝胶可以避免带子伪造。

It was confirmed via special handwriting that the three bands originated from the same membrane. By manipulating the total protein mass, we faked the low, high, and medium trends of protein bands, but the reference protein also showed the same trend, proving that there were traces of forgery. Red arrows indicate individual handwriting. The experimental procedure was the same as the standard experimental procedure.
通过特殊笔迹证实，这三条谱带来自同一膜。通过操纵总蛋白质量，我们伪造了蛋白质条带的低、高、中趋势，但参考蛋白也显示了相同的趋势，证明存在伪造的痕迹。红色箭头表示个人笔迹。实验程序与标准实验程序相同。

2.7. Protein-Free Rapid Blocking Buffer Outperforms Skim Milk
2.7.无蛋白快速封闭缓冲液性能优于脱脂牛奶

Blocking buffer prevents primary antibody non-specific binding and improves the signal-to-noise ratio. For some high-quality antibodies, blocking may not be performed. We compared the blocking efficiency of polyvinylpyrrolidone (PVP-40) 1% + Tween 20 0.05% with 5% skim milk + Tween 20 0.05%. The signal intensities of AKT, Rab27a, and CD81 were significantly higher in PVP-40 blocking for 10 min than skim milk blocking for 1 h, and the signal intensities of TSG101, KCa3.1, and GAPDH were also not inferior to those of the latter (Figure 5). In addition, commercial blocking buffer did not show overwhelming advantages compared with PVP-40 (Figure 1D). However, aqueous buffers of PVP-40 are prone to spoilage and can only be stored for a week at room temperature. Adding 0.05–1% non-toxic ProClin (Beyotime, Shanghai, China) can greatly extend the shelf life.
阻断缓冲液阻止了一抗的非特异性结合，提高了信噪比。对于一些高质量的抗体，可能不会进行阻断。比较了聚乙烯吡咯烷酮(PVP-40)1%+吐温20 0.05%和5%脱脂牛奶+吐温20 0.05%的封闭率。PVP-40阻断10min的AKT、Rab27a和CD81的信号强度显著高于阻断1h的脱脂牛奶，TSG101、KCa3.1和GAPDH的信号强度也不低于后者(图5)。此外，与PVP-40相比，商用阻塞缓冲区并没有显示出压倒性的优势(图1D)。然而，PVP-40的含水缓冲液容易变质，在室温下只能储存一周。添加0.05-1%的无毒普罗克林(上海碧瑶泰、中国)可大大延长保质期。

Figure 5. Comparison of the blocking efficiency of PVP-40 and skim milk.
图5.PVP-40和脱脂牛奶的阻隔效率比较。

The efficiency of PVP-40 blocking for 10 min was comparable to that of skim milk for 1 h. The experimental procedure was the same as the standard experimental procedure except for the blocking reagents (n = 6).
PVP-40封闭10min的效率与脱脂牛奶封闭1h的效率相当，除封闭试剂(n=6)外，实验程序与标准实验程序相同。

3. Discussion 3.讨论

Despite the importance of immunoblotting in biochemical research, the reproducibility of results is poor due to tedious operation and too many uncontrollable variables. To this end, we integrated the existing technologies and solutions to try to produce a guideline that enables beginners to quickly master this skill. Meanwhile, we tried to reduce the experimental cost as much as possible by improving the existing equipment and provided solutions to the current problems.
尽管免疫印迹在生化研究中具有重要意义，但由于操作繁琐，不可控变量太多，结果的重复性较差。为此，我们整合了现有的技术和解决方案，试图产生一种指导方针，使初学者能够快速掌握这项技能。同时，通过对现有设备的改进，尽可能地降低实验成本，并针对目前存在的问题提出了解决方案。

The concentration, solubility, and abundance of the target protein play a decisive role in the results of immunoblotting. It is necessary to quantify the protein concentration before proceeding to the next step. When the sample contains numerous cells, it is easy to make the sample viscous, which causes the tip of the pipette to be blocked and increases the sample loading error. The main reason for this stickiness is the release of nuclear DNA. Studies have shown that sonication can generate cleavage stress to break down the DNA structure, which dramatically improves the lysis and dissociates the target protein [14,15]. Of course, repeated grinding, pipetting, DNase, or dilution with RIPA and loading buffer (4:1) can also reduce viscosity.
目的蛋白的浓度、溶解度和丰度对免疫印迹的结果起决定性作用。在进行下一步之前，有必要对蛋白质浓度进行量化。当样品中含有许多细胞时，容易使样品变得粘稠，导致吸管尖端堵塞，增加样品加载误差。这种粘性的主要原因是核DNA的释放。研究表明，超声波可以产生切割应力来破坏DNA结构，这大大改善了裂解和解离目标蛋白[14，15]。当然，反复研磨、移液、脱氧核糖核酸酶或用RIPA和加载缓冲液(4：1)稀释也可以降低粘度。

Throughout, reagents are mixed one by one and poured into the gel-making glass plate. Mixing is time-consuming, labor-intensive, and easily confused. We optimized the gel preparation scheme by mixing the reagents into a system, which means that the preparation of the gel is shortened to 10 min. Increasing AP or TEMED by 50–100% significantly accelerates the gel solidification. However, excessive AP or TEMED causes gel burning and distortion of the bands during electrophoresis. The polymerization is fast under alkaline conditions, but the gel is hard and brittle when the alkalinity is too strong. AP or TEMED should be reduced when a high pH is needed.
整个过程中，试剂一一混合并倒入凝胶制作玻璃板中。混合耗时、费力且容易混淆。我们通过将试剂混合到系统中来优化凝胶制备方案，这意味着凝胶的制备缩短至10分钟。将AP或TEMED增加50-100%显着加速凝胶的凝固。然而，过量的AP或TEMED会导致凝胶燃烧和凝胶在凝胶过程中扭曲。碱性条件下聚合反应很快，但碱性太强时凝胶又硬又脆。当需要高pH值时，应降低AP或TEMED。

Traditional electrophoresis requires at least 90 min, which is too time-consuming for high-throughput experiments. Indeed, capillary electrophoresis has revolutionized this field due to its fast speed and high throughput [16,17]. However, it requires the purchase of expensive equipment and custom consumables. This will undoubtedly result in a reluctance to invest in stretched laboratories. Dumut’s laboratory developed a new electrophoresis buffer (100 mM Tris, 100 mM Tricine, and 100 mM HEPES) that could complete electrophoresis in 35 min. Additionally, the resolution of small-molecular-weight proteins could be improved [18]. However, Tricine is not cheap. Our self-developed electrophoresis buffer can significantly shorten the electrophoresis time and effectively separate proteins of different molecular weights. Because of the enhanced ionic strength, the heat increases significantly with the voltage, so the heat dissipation is particularly important. In addition, it is vital to maintain the pH of the electrophoresis buffer. Our results show that the difference in pH between the internal and external buffers after electrophoresis was about 0.3, showing a good buffering performance. Of course, we only tested the buffering capacity of the HEPES, and other “good” buffer systems should theoretically produce excellent results.
传统的电泳法至少需要90分钟，这对于高通量实验来说太耗时了。事实上，毛细管电泳因其快速和高通量而彻底改变了这一领域[16，17]。然而，它需要购买昂贵的设备和定制的消耗品。这无疑将导致不愿投资于捉襟见肘的实验室。杜穆特的实验室开发了一种新的电泳液(100 mm Tris、100 mm Tricine和100 mm HEPES)，可以在35分钟内完成电泳。此外，还可以提高小分子蛋白质的分辨率[18]。然而，Tricine并不便宜。我们自行研制的电泳液可以显著缩短电泳法的时间，有效分离不同分子量的蛋白质。由于离子强度的增强，热随着电压的升高而显著增加，因此散热就显得尤为重要。此外，保持电泳液的pH值也是至关重要的。结果表明，电泳后内外缓冲液的pH差约为0.3，表现出良好的缓冲性能。当然，我们只测试了HEPES的缓冲能力，其他“好”的缓冲系统理论上应该会产生很好的结果。

Compared with the traditional wet electrotransfer, the efficiency of the semi-dry system is unparalleled. Villanueva’s laboratory has successfully used isopropanol in Towbin’s transfer buffer [19]. Ghanshyam et al. tried to reduce the concentration of methanol. Their results suggested that methanol in electrotransfer buffer had little to no effect on large protein signals. However, a lower concentration of methanol (10%) was sufficient to produce a maximal signal for proteins with small or medium molecular weights [20]. Our research confirmed that ethanol is also reasonable for proteins with small or medium molecular weights. Proteins were efficiently transferred to both NC and PVDF membranes using an ethanol-based electrotransfer buffer. In addition, adding SDS increased the electrotransfer efficiency [21].
与传统的湿电转移相比，半干系统的效率无与伦比。维拉纽瓦的实验室已成功在Towbin的转移缓冲液中使用了丙酮[ 19]。Ghanshyam等人试图降低甲醇的浓度。他们的结果表明，电转移缓冲液中的甲醇对大蛋白质信号几乎没有影响。然而，较低浓度的甲醇（10%）足以为小分子或中等分子量的蛋白质产生最大信号[ 20]。我们的研究证实，乙醇对于小分子或中等分子量的蛋白质也是合理的。使用乙醇基电转移缓冲液将蛋白质有效转移到NC和氨纶膜上。此外，添加十二烷基硫酸钠还提高了电转移效率[ 21]。

Garic et al. developed an electrotransfer buffer (48 mM Tris, 20 mM HEPES, 1.3 mM NaHSO₃, 1.0 mM EDTA, and 1.3 mM N, N-dimethylformamide) that could be used to complete the electrotransfer in 12 min. NaHSO₃ compensates for the lack of SDS by acting as a reducing agent, enhances the solubility of large proteins, and acts as a scavenger for free radicals produced by HEPES and HEPPS/EPPS. Furthermore, EDTA indirectly stabilizes piperazine-ring-containing buffers and chelates metal ions. N, N-dimethylformamide acts as a chaotropic agent [22]. Subsequently, Grogery and colleagues filed a patent (US9989493B2) for a rapid electrotransfer buffer (336 mM Tris, 260 mM Glycine, 140 mM Tricine, and 2.5 mM EDTA) that could transfer 10-300 kDa proteins in 5–10 min. This rapid electrotransfer buffer, which is 10 times the ionic strength of traditional transfer buffer, generates considerable heat. Therefore, adding 20% ethanol is effective in dissipating heat.
Garic等人开发了一种电转移缓冲液（48 mMTris、20 mMMHEPES、1.3 mMMNaHSO、1.0 mMEDTA和1.3 mMMN，N-二甲基甲氨），可用于在12分钟内完成电转移。NaHSO通过充当还原剂来弥补了SDs的缺乏，增强了大蛋白质的溶解度，并充当HEPES和HEPPS/EPPS产生的自由基的清除剂。此外，EDTA还间接稳定含嗪环的缓冲液并可以水合金属离子。N，N-二甲基甲胺充当离液剂[ 22]。随后，Grogery及其同事申请了一项快速电转移缓冲液（336 mM Trisis、260 mM Glycine、140 mM Tricine和2.5 mM EDTA）的专利（US 9989493 B2），可以在5-10分钟内转移10-300 KDa蛋白质。这种快速电转移缓冲液的离子强度是传统转移缓冲液的10倍，会产生相当大的热量。因此，加入20%的乙醇对于散热是有效的。

Interestingly, the pre-stained protein marker could not truly reflect whether the protein was transferred to the 0.45 μm PVDF membrane or not. In the semi-dry system, dye can be transferred to the 0.45 μm PVDF membrane and the lower filter paper simultaneously within 5 min, but almost no protein is transferred to 0.45 μm PVDF membrane. In fact, pre-stained protein markers are mixtures of purified proteins and dyes, which may separate when exposed to an electric field. Hence, whether proteins are transferred to membranes is time-dependent.
有趣的是，预染色的蛋白标记物并不能真正反映蛋白是否转移到0.45 μm的聚偏氟乙烯膜上。在半干体系中，染料可以在5分钟内同时转移到0.45 μm的聚偏氟乙烯膜和下纸片上，但几乎没有蛋白质转移到0.45 μm的聚偏氟乙烯膜上。事实上，预染色的蛋白质标记物是纯化的蛋白质和染料的混合物，当暴露于电场时可能会分离。因此，蛋白质是否转移到膜上是取决于时间的。

Hitherto, academia has still been unable to reconcile the transfer problems of proteins with different molecular weights. Small-molecular-weight proteins move faster than large-molecular-weight proteins, which leads to an asynchronous transfer. The traditional practice transfers proteins of large and small molecular weight separately. In-depth discussions of the reason for this are still taking place. The 0.45 μm PVDF membrane may have a weak retention of fast-moving small-molecular-weight proteins, which can easily penetrate the membrane. Karey et al. reported that using 0.5% glutaraldehyde for the detection of low-molecular-weight acidic and basic isoelectric point proteins increased sensitivity by 1.5-12-fold in immunoblotting [23]. Jing and colleagues proposed that organic solvents and heating substantially avoided the loss of protein signals [24]. Taken together, their methods could lower the detection threshold by enhancing protein binding to membranes. However, the signal enhancement of this fixation method can only be limited to the detection of proteins with a certain molecular weight span. Thus, we propose the following solutions: 1. Reduce the voltage and prolong the electrotransfer time. When the electric field force and the resistance of the PVDF membrane (mechanical resistance and electrostatic force) reach a balance, the protein will not move directionally. 2. Use multiple 0.45 μm PVDF membranes to intercept the transferred protein. When the protein transferred the first 0.45 μm PVDF membrane, the second membrane intercepted the first transferred small-molecular-weight protein within a certain period of time. 3. Reducing the pore size of the PVDF membrane means increasing the resistance. If a 0.22 μm PVDF membrane is used, an extended blocking time may solve the problem of a deep background. However, these solutions can only improve the ability to retain proteins with different molecular weights to a certain extent. Increasing membrane thickness or the stacking of membranes with different pore sizes may provide some new ideas.
到目前为止，学术界仍然无法协调不同分子量蛋白质的转移问题。小分子量蛋白质比大分子量蛋白质移动得更快，这导致了一种异步转移。传统的做法是将大分子和小分子的蛋白质分开转移。对其原因的深入讨论仍在进行中。0.45μm聚偏氟乙烯膜对快速运动的小分子蛋白质有较弱的截留力，容易透过膜。Karey等人。报道称，使用0.5%的戊二醛来检测低分子量的酸性和碱性等电点蛋白，使免疫印迹的灵敏度提高了1.5-12倍[23]。Jing和他的同事提出，有机溶剂和加热大大避免了蛋白质信号的损失[24]。总而言之，他们的方法可以通过增强蛋白质与膜的结合来降低检测阈值。然而，这种固定方法的信号增强只能限于对具有一定分子量区间的蛋白质的检测。因此，我们提出了以下解决方案：1.降低电压，延长电传输时间。当电场力和PVDF膜的电阻(机械阻力和静电力)达到平衡时，蛋白质不会定向运动。2.用多层0.45μm聚偏氟乙烯膜截留转运蛋白。当蛋白转移到第一个0.45μm聚偏氟乙烯膜时，第二个膜在一定时间内截留第一个转移的小分子蛋白。3.减小PVDF膜的孔径意味着增加膜的阻力。如果是0。使用22 μm的聚偏氟乙烯膜，延长封闭时间可以解决深背景的问题。但这些溶液只能在一定程度上提高保留不同分子量蛋白质的能力。增加膜厚度或堆叠不同孔尺寸的膜可能会提供一些新的想法。

The problem that the reference protein and the target protein are not visualized on the same membrane is an inherent defect of immunoblotting. Without the reference protein, these trends can be manipulated at will. Of course, the most effective way is to incubate the same membrane after the primary antibody stripping. However, protein- or antibody-binding properties vary, so stripping conditions need to be explored, which undoubtedly consumes time and effort. This problem can be partly solved using the photography we proposed above. In addition, whole-cell proteins that can be used as reference proteins include GAPDH (37 kDa), Actin (42 kDa), α-Tubulin (50 kDa), β-Tubulin (55 kDa), and HSP90 (90 kDa) [25,26]. When target proteins of different molecular weight needs are cut on the same membrane, the reference protein can be cut in other uncut regions to ensure that each membrane has a reference protein.
参考蛋白和目标蛋白不在同一膜上可见的问题是免疫印记的固有缺陷。如果没有参考蛋白，这些趋势就可以随意操纵。当然，最有效的方法是在一抗剥离后温育同一膜。然而，蛋白质或抗体的结合特性各不相同，因此需要探索剥离条件，这无疑会消耗时间和精力。这个问题可以使用我们上面提出的摄影技术部分解决。此外，可用作参考蛋白的全细胞蛋白包括GADH（37 KDa）、肌动蛋白（42 KDa）、a-Tubulin（50 KDa）、β-Tubulin（55 KDa）和HSP 90（90 KDa）[ 25，26]。当在同一膜上切割不同分子量需求的目标蛋白时，可以在其他未切割区域切割参考蛋白，以确保每个膜都有参考蛋白。

Blocking is an effective way to improve the signal-to-noise ratio. Skim milk needs to be prepared immediately because the solution deteriorates rapidly and disguises some primary antibodies. Other substances, such as bovine serum albumin, fish gelatin, and Tween-20, do not make the blocking time shorter [27]. Previous studies found that soymilk is an inexpensive alternative to the commercially available rapid blocking reagent [28]. However, it also possesses the drawback of not being suitable for long-term preservation. PVP-40, which has the properties of nontoxicity and biocompatibility, was reported as a blocking reagent as early as 30 years ago [29]. Cui reported that PVP has the advantage of no or very-low autofluorescence in any of the detection channels. However, it is less effective at blocking non-specific bands [30]. Through a series of time-gradient comparisons, we found that PVP-40 had a good blocking effect even over a short time period. The commercial blocking reagents may not offer a cost-effective advantage, as is shown in Figure 1D.
块效应是提高信噪比的一种有效方法。脱脂牛奶需要立即制备，因为这种溶液会迅速变质，并掩盖一些主要抗体。其他物质，如牛血清白蛋白、鱼胶和吐温-20，不会缩短封闭时间[27]。以前的研究发现，豆浆是一种廉价的替代商业上可获得的快速封闭试剂[28]。然而，它也有不适合长期保存的缺点。PVP-40具有无毒和生物相容性，早在30年前就被报道为封闭剂[29]。崔报告说，PVP的优势是在任何检测通道中都没有或非常低的自发荧光。然而，它在阻止非特定条带方面效果较差[30]。通过一系列的时间梯度比较，我们发现PVP-40即使在短时间内也具有良好的封闭效果。如图1D所示，商业封闭剂可能不会提供成本效益优势。

4. Conclusions 4.结论

We substantially modified the key steps of immunoblotting. First, the simplified gel preparation scheme dramatically increases speed and reduces the chance of failure. Second, the modified electrophoresis buffer formula drastically reduces the time taken while rivalling the traditional formula. Third, replacing methanol with ethanol in the electrotransfer buffer significantly reduces the exposure of laboratory operators to hazardous gases. Finally, PVP-40 achieves similar results while compensating for the shortcomings of skim milk. Compared with the traditional protocol, our standard experimental process improved efficiency by almost four-fold. Our modifications show that even low-quality antibodies can be visualized normally. If the primary antibody is incubated with the membrane at room temperature, the experiment can be completed within one day.
我们大幅修改了免疫印记的关键步骤。首先，简化的凝胶制备方案大大提高了速度并减少了失败的机会。其次，改进的凝胶缓冲液配方在与传统配方相媲美的同时大幅减少了所需的时间。第三，用乙醇取代电转移缓冲液中的甲醇可以显着减少实验室操作员接触危险气体的情况。最后，VP-40在弥补脱脂奶的缺点的同时也达到了类似的结果。与传统协议相比，我们的标准实验过程将效率提高了近四倍。我们的修改表明，即使是低质量的抗体也可以正常可视化。如果将一抗与膜在室温下孵育，则可以在一天内完成实验。

5. Materials and Methods
5.材料和方法

5.1. Reagents 5.1.试剂

Radio Immunoprecipitation Assay Lysis Buffer (RIPA, Servicebio, Wuhan, China), cocktail 100× (100 M PMSF (Beyotime, Shanghai, China), 1 mg/mL leupeptin (Beyotime, Shanghai, China), 1 mg/mL astatin (Beyotime, Shanghai, China)), loading buffer 5× ((1 M Tris 1.25 mL, sodium dodecyl sulfate (SDS) 0.5 g, bromophenol blue 25 mg, 100% glycerol 2.5 mL, β-mercaptoethanol 250 µL) add deionized water to 10 mL), 30% Acr-Bis (acrylamide: bisacrylamide = 29:1), ammonium persulfate (AP, Servicebio, Wuhan, China), tetramethylethylenediamine (TEMED, Servicebio, Wuhan, China), glycine (Biosharp, Beijing, China), Tris (Biosharp, Beijing, China), and 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES, Biofroxx, Einhausen, Germany). Primary antibodies: Cytochrome C (CyC, 12 KDa, Genetex, Irvine, CA, USA, 1:1000), CD81 (exosome membrane marker, 26 KDa, Abmart, Shanghai, China, 1:1000), Rab27a (exosome secretion-related protein, 28 kDa, Servicebio, Wuhan, China, 1:1000), GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase, 36 KDa, Servicebio, Wuhan, China, 1:1000), TSG101 (Tumor Susceptibility Gene 101 Protein, 46 kDa, Servicebio, Wuhan, China, 1:1000), KCa3.1 (Potassium Calcium-Activated Channel Subfamily N Member 4, 48 kDa, Proteintech, Wuhan, China, 1:1000), AKT (AKT Serine/Threonine Kinase 1, 60 kDa, Servicebio, Wuhan, China, 1:1000), and PINK (PTEN-Induced Putative Kinase Protein, 63 kDa, Genetex, Irvine, CA, USA, 1:1000). Secondary horseradish peroxidase-conjugated antibody (Proteintech, Wuhan, China, 1:3000), pre-stained protein marker (ThermoFisher, Boston, MA, USA, 26616), 0.22 µm and 0.45 µm polyvinylidene fluoride membrane (PVDF, Millipore, Burlington, MA, USA), 0.45 µm nitrocellulose membrane (NC, Millipore, Burlington, MA, USA), PBS (Biosharp, Beijing, China), Tween-20 (Servicebio, Wuhan, China), and ECL Chemiluminescence Kit (Servicebio, Wuhan, China).
放射免疫沉淀分析裂解缓冲液(利华生物，武汉，中国)，鸡尾酒100×(100M PMSF(百时美施贵宝，上海，中国)，1毫克/毫升亮肽(百时美施贵宝，上海，中国)，1毫克/毫升他汀(百时美施贵宝，上海，中国))，负载缓冲液5×(1M Tris 1.25毫升，十二烷基硫酸钠0.5g，溴酚蓝25毫克，100%甘油2.5，β-巯基乙醇250微克L)，加入去离子水至10毫升)，30%ACR-BIS(丙烯酰胺：双丙烯酰胺=29：1)，过硫酸铵(AP，服务生物，武汉，过硫铵)甘氨酸(北京，中国)、三元(北京，中国)和2-[4-(2-羟乙基)哌嗪-1-基]乙磺酸(HEPES，生物弗罗克斯，德国艾因豪森)。一抗：细胞色素C(CyC，12 KDa，GeneTex，美国欧文，CA，1：1000)，CD81(胞外体膜标记物，26 KDa，Abmart，上海，中国，1：1000)，Rab27a(外体分泌相关蛋白，28 kDa，ServiceBio，武汉，中国，1：1000)，GAPDH(甘油醛-3-磷酸脱氢酶，36 KDa，ServiceBio，武汉，中国，1：1000)，TSG101(肿瘤易感基因101蛋白，46 kDa，ServiceBio，武汉，中国，1：1000)，KCa3.1(钾激活钙通道亚家族N成员4，48 kDa，Da Proteintech，武汉，中国，1：1000)AKT(AKT Serine/Threonine Kinase 1，60 kDa，武汉，中国，1：1000)和PINK(PTEN诱导的可能的Kinase蛋白，63 kDa，GeneTex，美国，欧文，CA，1：1000)。二次辣根过氧化物酶标记抗体(Proteintech，武汉，中国，1：3 000)，预染蛋白标记物(ThermoFisher，波士顿，美国，26616)，0.22和0.45µm聚偏氟乙烯膜(聚偏氟乙烯，微孔，美国伯灵顿，美国)，0。45 µm硝酸纤维素膜（NC，Millipore，Burlington，MA，USA）、PBS（BioSharp，中国北京）、Tween-20（Servebio，中国武汉）和MEL化学发光试剂盒（Servebio，中国武汉）。

5.2. Tissue Lysates 5.2.组织裂解物

This study was approved by the Animal Studies Subcommittee of our Institutional Review Board. We extracted proteins from 30 mg of C57BL/6J mouse myocardial tissue, adding 800 μL RIPA and 100× cocktail. Then, the tissue was ground twice (JingXin, shanghai, China; 10 Hz, 45 s) and lysed on ice for 10 min. The tissue suspension was centrifuged at 12,000 g for 20 min and the sediment was discarded. The protein concentration was determined by a BCA Protein Assay Kit (Aspen, Wuhan, China), according to the manufacturer’s instructions. The sample was added to 5× loading buffer and boiled at 100 °C for 10 min.
这项研究得到了我们机构审查委员会动物研究小组委员会的批准。从30 mg C57BL/6J小鼠心肌组织中提取蛋白质，加入800μL RIPA和100×鸡尾酒。然后，将组织粉碎两次(静心、上海、中国；10赫兹，45 S)，在冰上裂解10分钟。组织悬浮液在12,000g下离心20分钟，并去掉沉淀物。根据制造商的说明，用BCA蛋白质分析试剂盒(Aspen，武汉，中国)测定蛋白质浓度。将样品加入5倍加载缓冲液中，在100℃下煮沸10min。

5.3. Standard Experimental Procedure
5.3.标准实验程序

A total of 30 µg per well of total protein was separated by quick gel (Mini Gel). Electrophoresis was performed with the modified electrophoresis running buffer at 200 V for 35 min (Mini-PROTEAN Tetra Cell Systems, Bio-Rad, Los Angeles, CA, USA). Then, proteins were transferred to a 0.45 µm PVDF membrane with ethanol-based electrotransfer buffer with SDS for 25 min (Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell, Bio-Rad, Los Angeles, CA, USA) and blocked with PVP-40 for 10 min. Subsequently, the membrane was incubated with the primary antibody overnight at 4 °C. The membrane was then rinsed three times in PBST. Membranes were incubated with the corresponding secondary antibodies at room temperature for 1 h, rinsed three times with PBST, and then developed (ChemiDoc XRS System, Bio-Rad, Los Angeles, CA, USA).
用快速凝胶(Mini Gel)分离每孔总蛋白30微克。用改良的200V运行缓冲液进行电泳35min(Mini-Protean Tetra Cell Systems，Bio-Rad，洛杉矶，加利福尼亚州，美国)。然后，将蛋白质转移到含有十二烷基硫酸钠的乙醇基电转移缓冲液的0.45微米PVDF膜上25分钟(美国加利福尼亚州洛杉矶，Bio-Rad的Trans-Blot SD半干凝胶转移细胞)，并用PVP-40阻断10分钟。随后，将膜与一抗在4℃下孵育过夜，然后在PBST中冲洗三次。膜与相应的二抗在室温下孵育1h，用PBST漂洗3次，然后显影(ChemiDoc XRS System，Bio-Rad，洛杉矶，加利福尼亚州，美国)。

5.4. Analysis of Signal Intensities
5.4.信号强度分析

The signal intensity of the protein bands was measured and compared using Image Lab (Bio-Rad, Hercules, CA, USA) and GraphPad Prism 8.0 (GraphPad Software, Boston, MA, USA). The density volume within each protein band was measured as intensity/mm². All values are means ± S.E.
使用Image Lab(Bio-Rad，Hercules，CA，USA)和GraphPad Prism 8.0(GraphPad Software，Boston，MA，USA)测量和比较蛋白质带的信号强度。每条蛋白条带内的密度体积以强度/mm ² 表示。所有值均为平均值±S.E.