蛋白质与蛋白组学

Light scattering from either turbid protein samples or particles suspended in the sample with a comparable size to the incident wavelength (250– 300 nm) can reduce the amount of light reaching the detector leading to an increase in apparent absorbance. Filtration using 0.2 um filter units (that do not adsorb proteins), or centrifugation can be performed prior to analysis to reduce light scattering. Corrections for light scattering can be performed by measuring absorbance at lower energies (320, 325, 330, 335, 340, 345, and 350 nm), assuming the protein does not display significant absorbance at these wavelengths. A log–log plot of absorbance versus wavelength should generate a linear response that can be extrapolated back to 280 nm, the resulting antilog of which will give the scattering contribution at this wavelength (Leach and Scheraga, 1960).
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浑浊样品在测定前要离心或过滤，自然。
用320-350nm处的吸收来消除280nm的散射吸收？有局限性。万一样品中有某些物质在此波长范围内有特异性的吸收呢？

Nucleic acids absorb strongly at 280 nm and are a common contaminant of protein preparations. A pure protein preparation is estimated to give a ratio of A280 to A260 of 2.0 while, if nucleic acid is present, the protein concentration can be derived by the following formula (Groves et al., 1968).
Protein concentration （mg/ml）=1.55A280—0.76A260

核酸在280nm有强吸收，而且是蛋白质制备过程中的常见污染物。制备好的纯蛋白，其A280/A260大约为2.0。如果有核酸存在，蛋白浓度可以用下式计算：
蛋白浓度（mg/ml）=1.55A280—0.76A260

这个公式我也比较喜欢用。可以推导一下，如果是纯蛋白：
蛋白浓度（mg/ml）=1.55A280—0.76A260=（1.55—0.76/2)A280=1.17 A280

3.4. Ultraviolet absorbance at 205 nm (Range: 1–100 ug)
The peptide bond absorbs photons at a maximum wavelength below 210 nm. However, the broad absorption peak of the peptide bond allowsmeasurements at longer wavelengths, which can have many practical advantages in terms of instrumentation andmeasurement accuracy.Due to interference fromsolvents and components of biological buffers, absorbance at 214 and 220 nm is often used as an alternative to measure proteins and peptides.

紫外吸收205nm（范围：1-100ug）

肽键吸收光子的最大波长在210nm以下。然而，肽键宽广的吸收峰容许检测在更大的波长处进行，这一点对设备和检测的精度有很多实际益处。由于生物缓冲液中溶剂和其他组分的干扰，214nm和220nm的吸收值常用来作为检测蛋白和多肽的替代波长。

The large number of peptide bonds within proteins can make Abs205 nm measurements more sensitive and display less protein-to-protein variability than Abs280 nm measurements. Most proteins have extinction coefficients at Abs205 nm for a 1-mg/ml solution of between 30 and 35; however, an improved estimate can be obtained using Eq. (8.3) that takes into account variations in tryptophan and tyrosine content of the protein to be quantitated (Scopes, 1974). Absorbance at 205 nm is used to quantitate dilute solutions, or for short path length applications, for example, continuous measurement in column chromatography, or for analysis of peptides where there are few, if any aromatic amino acids.


蛋白质中大量肽键的存在使得A205nm比A280nm检测更敏感，而且表现出更小的蛋白个体差异。多数蛋白其1mg/ml溶液的A205nm消光系数为30-35之间；考虑到待定量蛋白质中色氨酸和酪氨酸的含量差异，下面的方程可以给予一个改进后的估值。205nm吸收可用来定量稀蛋白溶液；或在短光径处应用，例如柱层析中的连续检测；或用于芳香性氨基酸含量较少的多肽分析。

平时很少很少用205-215nm这段波长去做在线监测，如果蛋白浓度低到这个程度，我往往是拒绝做实验了，呵呵。

3.5. Calculation of the extinction coefficient
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4. Dye-Based Protein Assays
蛋白质的染料分析方法
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5. Coomassie Blue (Bradford) Protein Assay (Range: 1–50 ug)
The Bradford assay encompasses various preparations of the dye Coomassie Brilliant Blue G-250 used for protein quantitation purposes, and was first described by Bradford (1976). The basic mechanism of the assay is the binding of the dye at acidic pH to arginine, histidine, phenylalanine, tryptophan and tyrosine residues (de Moreno et al., 1986), and hydrophobic interactions (Fountoulakis et al., 1992). The exact mechanism is however still not fully understood (Sapan et al., 1999). Upon binding protein, a metachromatic shift from 465 to 595 nm is observed due to stabilization of the anionic form of the dye. The majority of the observed signal is due to the interaction with arginine residues, resulting in the wide protein to protein variation characteristic of Bradford assays (Fig. 8.3).

考马斯亮蓝（Bradford）法蛋白质分析（范围：1-50ug）
Bradford分析法由Bradford于1976年首先提出，它包含了各种各样用于蛋白质定量的考马斯亮蓝G-250染料的制备。本方法的基本原理是染料在酸性pH条件下与精氨酸、组氨酸、苯丙氨酸、色氨酸和酪氨酸的结合作用(de Moreno et al., 1986)，以及疏水作用(Fountoulakis et al., 1992)。确切的原理依然还没有完全了解。由于结合蛋白的作用，染料的阴离子形式被固定化，可以观察到465nm到595nm的光染迁移。观察到的信号主要是由于染料与精氨酸残基的作用，所以在Bradford分析法中有着广泛的蛋白质和蛋白质的各种个性差异。

原理我还真没有看过。稍稍分析了一下。精氨酸和组氨酸是碱性氨基酸，在低pH条件下带正电荷，结合染料的阴离子形式；苯丙氨酸、色氨酸和酪氨酸是芳香性氨基酸，带有苯环，与染料以疏水方式结合。有点儿迷惑的是，为什么没有赖氨酸这个碱性氨基酸？

5.1. Reagents
Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml of 95% ethanol and add 100 ml of 85% phosphoric acid while stirring continuously. When the dye has dissolved dilute to 1 l in water. The reagent is stable for up to a month at room temperature; however, for long-term storage keep at 4C, if precipitation occurs filter before use.

试剂
100mg考马斯亮蓝G-250溶于50ml 95%的乙醇中，再搅拌连续加入100ml 85%的磷酸。染料溶解完毕后，用水稀释至1L。室温下，本试剂可以稳定一个月；但是长期储存应置于4度，有沉淀时在使用前过滤。

有沉淀过滤后可以使用，说明对本试剂的配制来说要求不是很精确，或者说方法本身有误差。

5.2. Procedure
1. Prepare standards in the range 100–1500 ug/ml in a Bradford-compatible buffer. For more dilute samples the sensitivity can be extended by increasing the ratio of sample to reagent volumes (Micro Bradford assay: 1–25ug/ml). If the ratio of the sample to dye is too high, the pH of the reaction mixture could increase leading to higher background responses.

步骤
在Bradford法兼容的缓冲液中配制100–1500 ug/ml范围的标准蛋白。对低浓度样品，灵敏度可由增加样品与试剂的体积比来提高（Micro Bradford assay: 1–25ug/ml）。如果样品与染料的比例过高，反应混合物的pH可能会上升，而导致较高的背景值。

2. Add the standard and unknown samples to disposable cuvettes (plastic disposable cuvettes and microplates should be used as the dye sticks to various surfaces).

加入标准液和未知样品液到一次性的比色皿中（染料会粘附于各种容器的表面，所以要用塑料一次性比色皿和微孔板）

3. Allow the Bradford reagent to warm to room temperature. Add 1 ml of the dye solution to 25 ml of the protein sample, mix and incubate for 10 min at room temperature.

Bradford试剂平衡至室温。25ul蛋白质样品中加入1ml染料溶液，室温混合孵育10min。

4. Measure the absorbance at 450 and 595 nm (for filter-based instruments a range from 570 to 610 nm can be used without significant loss of assay performance).

测量450和595nm的吸光度（对用滤光片的设备来说，570-610nm都能应用，而不会造成分析结果的重大偏差）。

5. Plot either the 595 nm data or for improved precision at lower response values the ratio 595 nm/450 nm. The standard response curve can be fit to a polynomial response, from which unknown protein estimates can be calculated.

用595nm的数据作图，或为了提高低浓度样品的检测精度，用595nm/450nm的数据作图。标准曲线可能符合多项式拟合，从而计算出未知蛋白样品的浓度估值。

5.3. Comments
The advantages of the Bradford assay include the ease of use, sensitivity and low cost of the reagents. For microplate-based assays the reagent volumes can be decreased giving a total volume of 300 ul. Due to the path of the light source on the majority of microplate spectrophotometers, it is recommended to use commercial sources of Bradford reagent that are less predisposed to precipitation during prolonged storage.

评价
Bradford分析法的优点包括应用方便、灵敏度好，试剂费用低。用微孔板进行的分析，试剂的体积可以减小到300ul的总体积。由于多数微孔板分光光度计光源路径的原因，推荐使用商品化的Bradford试剂，这样的试剂在较长的储存期内不容易析出沉淀。

解释一下。一般的分光光度计光的方向是从比色皿的侧面透过，所以试剂有微量的沉淀可能对检测影响不大。微孔板的光方向是由上到下的垂直方向，沉淀将极大地影响光吸收值的读数。

We have observed significant variation in response between various commercial suppliers of Bradford preparations (Noble et al., 2007). This appears to be most pronounced when analyzing low-molecular-weight proteins or peptides. Indeed the assay is reported to display a molecular weight cutoff ‘‘threshold’’; requiring a certain number of residues for full signal development (de Moreno et al., 1986). Changes in the formulation of the Bradford reagent are reported to change the response generated from specific proteins; therefore, care should be taken when comparing Bradford data from different suppliers or preparations (Chan et al., 1995; Friedenauer and Berlet, 1989; Lopez et al., 1993; Read and Northcote, 1981).

在不同商品化Bradford试剂供应商之间，我们观察到检测值的很大差异。在分析低分子量蛋白或多肽时，这种现象最为常见。确实，本分析方法据报道，有个分子量的限制“阀值”；需要一定数量的氨基酸残基来分析全长的蛋白。Bradford试剂配方的改变据报道会改变特定蛋白所产生的检测值；因此，比较不同供应商的Bradford数据时要小心。

The Bradford assay is sensitive to interferences from various reagents detailed in Table 8.1 that include most ionic and nonionic detergents and glycosylated proteins. If precipitation of the reaction mixture occurs, for example hydrophobic or membrane proteins, the reaction can be supplemented with 1 M NaOH at 5–10% (v/v) to aid solubilization.

Bradford分析法对很多种试剂的干扰敏感，详见表8.1，它包括多数离子型和非离子型表面活性剂，以及糖基化的蛋白质。如果反应混合物有沉淀产生，例如有些疏水性蛋白或膜蛋白，反应可以添加5-10%（v/v）体积比的1M NaOH 以增加溶解性。

好奇一下。试剂是100ml 85%的磷酸，用水稀释至1L，即磷酸浓度为8.5%，其摩尔浓度大约为1.5M。25ul蛋白质样品中加入1ml染料溶液，对磷酸浓度的影响可以忽略。这样，添加5-10%（v/v）体积比的1M NaOH应该对体系的pH没有根本性的影响吧，如何又能促进沉淀蛋白的溶解呢？

The Lowry protein assay is sensitive to many interfering compounds (Table 8.1), which may not generate a linear response (making extrapolations of interfering data complex). Formation of precipitates can occur with detergents, lipids, potassium ions, and sodium phosphate.

Lowry蛋白质分析方法对很多干扰性的化合物敏感，见表8.1，这可以导致非线性的反应结果。有表面活性剂、脂类、钾离子和磷酸钠存在时，可能形成沉淀物。

7. Bicinchoninic Acid (BCA) (Range: 0.2–50 ug)
The BCA assay replaces the Folin–Cioalteu’s reagent as described for the Lowry method with Bicinchoninic acid that results in a protein assay with improved sensitivity and tolerance to interfering compounds (Smith et al., 1985). The BCA reaction forms an intense purple complex with cuprous ions (Cut) resulting from the reaction of protein and alkaline Cu2t. The residues that contribute to the reduction of Cu2t include the cysteine, cystine, tryptophan, tyrosine, and the peptide bonds (Smith et al., 1985). The chemical reaction is temperature dependent with different functional groups displaying a different reactivity at elevated temperatures, which result in less protein variability (Wiechelman et al., 1988). At elevated temperatures (60C compared to 37C), more color formation is observed due to the higher reactivity of tryptophan, tyrosine, and peptide bonds.

BCA分析法(范围: 0.2–50 ug)
BCA法是用二喹啉甲酸替代了Lowry中所用的Folin酚试剂，提高了蛋白质分析的灵敏度和对干扰物质的耐受能力。蛋白质与碱性二价铜离子反应生成一价铜离子，此一价铜离子在BCA反应中形成一种紧密的紫色复合物。对二价铜离子还原反应有贡献的氨基酸残基有半胱氨酸、胱氨酸、色氨酸、酪氨酸和肽键。本化学反应与不同功能基团之间是温度依赖性关系，当升高温度时表现出不同的反应程度，从而对蛋白质的变化不敏感。升高温度（37度到60度），由于与色氨酸、酪氨酸和肽键的反应程度提高，可以观察到更多的颜色物质形成。

后一部分有些拗口，解释一下。
1、BCA反应与蛋白质中不同的氨基酸残基反应程度不同，
2、由于蛋白质氨基酸组成的差异，可能会造成同样浓度的不同蛋白质其紫色复合物形成的差异。
3、BCA反应随温度的升高其反应程度提高，紫色复合物形成提高。
4、提高BCA反应的温度，可以减小由蛋白质氨基酸差异所带来的颜色差异。

Most of the commercial preparations are formulated close to the original preparation described by Smith et al. (1985), which is described in the following subsections. Variations have been employed to improve the sensitivity of the assay and can be obtained from commercial sources (Pierce, Novagen). The sample-to-working reagent ratio can be varied to maximize signal, or reduce assay interference, typically ratios of 8–20-fold excess of BCA working reagent are added to the protein sample.

大部分商品化的BCA试剂配方与原始文献Smith等人的制备方法相近，下面的小节将做描述。商品化来源的试剂（Pierce, Novagen）其改变是为了提高分析的灵敏度。样品与工作试剂的比例变化可以用来提高检测信号值，或用来减少分析干扰，典型地，8-20倍BCA工作试剂加入到蛋白质样品中。

7.1. Reagents
Reagent A: 1 g sodium bicinchoninate, 2 g Na2CO3, 0.16 g sodium tartrate, 0.4 g NaOH, and 0.95 g NaHCO3, made up to 100 ml and the pH adjusted to 11.25 with either solid or concentrated NaOH.

试剂
A试剂：1g二喹啉甲酸钠，2g碳酸钠，0.16g酒石酸钠，0.4g氢氧化钠，和0.95g碳酸氢钠，配制为100ml溶液，用固体氢氧化钠或高浓度氢氧化钠溶液调节pH为11.25。

这个地方有些小问题。
1、没有讲化学药品是否含结晶水。这样的配方是不严密的。例如酒石酸钠有二水合的。
2、配制试剂应该先溶解化学药品，大约为80%的体积，然后调节pH，再定容至100%体积。
3、pH为11.25不知道该如何去调节？我们应该知道pH计在这个值是不准确的了。