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Introduction

Hoang Ha | Applications Scientist | Molecular Devices

Nucleic acid and protein quantitation are essential measurements upstream of many sophisticated assays in genetics and molecular biology. Various methods have been developed to quantitate these biological components, however, the most commonly used technique is still ultra-violet (UV) spectrophotometry. The basis of spectrophotometry is that every molecule absorbs or transmits light over a certain wavelength range, and their concentrations can be calculated by using the Beer-Lambert law (Equation 1) with preceding knowledge of the sample’s molar extinction coefficient and the measurement pathlength.

A = εcL

Equation 1: The Beer Lambert law states that absorbance (A) is equal to the measured molecule’s molar coefficient (ε) multiplied by the concentration (c) and pathlength (L) used to measure said molecule. Re-arranging the equation allows us to use absorbance to calculate concentration.

Nucleic acid quantitation is a very well-established technique, and its basic principles have not been modified much since its inception. To calculate nucleic acid concentration, samples are measured at 260 nm, and auxiliary measurements are taken at 230 nm and 280 nm wavelengths to check for sample purity.

Protein samples can be quantitated using UV spectrophotometry as well, however, there are more accurate colorimetric assays available. The UV spectrophotometric method utilizes tryptophan’s aromatic properties to absorb light at 280 nm, but calculated protein concentrations can be skewed by varying amounts of tryptophan residues in the amino acid sequence. Alternatively, the Bicinchoninic Acid (BCA) protein assay can measure protein concentrations independent of amino acid sequence and length. The assay utilizes the copper-based Biuret reaction where the amino acid backbone forms a color-chelate complex with copper molecules in an alkaline environment.

The SpectraMax® ABS Plus is a compact UV-Vis absorbance microplate reader ideal for these types of quantitative assays. Here, we demonstrate several different ways the ABS Plus reader, in combination with the SpectraDrop™ Micro-Volume Microplate and SoftMax® Pro Software, can quantitate double-stranded DNA and bovine serum albumin (BSA) protein.

Materials

• SpectraMax ABS Plus Microplate Reader (Molecular Devices cat. #ABS Plus)
• SpectraDrop Micro-Volume Microplate (Molecular Devices cat. #0200-6262)
• 96-well, clear, flat-bottom polystyrene microplate (Greiner Bio-One cat. #655101)
• UltraPure ^™ Calf Thymus DNA Solution (ThermoFisher Scientific cat. #15633019)
• Pierce BCA Protein Assay Kit (ThermoFisher Scientific cat. #23225)
• Pierce ^™ Bovine Serum Albumin Standard Ampules, 2 mg/mL (ThermoFisher Scientific cat. #23209)
• UV-Star ^® 96-well microplates (Greiner Bio-One cat. #655801)

Methods

DNA quantitation

A 2-fold dilution series of dsDNA starting at 1000 ng/µL was prepared from UltraPure calf thymus DNA diluted in 1x PBS. Either 2 µL or 4 µL of sample was pipetted onto a 64-well SpectraDrop microplate, and a 0.5 mm or a 1.0 mm coverslip was used to cover the dsDNA sample respectively. The preconfigured protocol, "SpectraDrop DNA Quantitation", was opened in SoftMax Pro Software, and the plate was read using the provided settings. A log-log curve fit was applied to the data, and a standard curve was generated using SoftMax Pro Software. Additionally, a spectral scan ranging from 220 nm to 350 nm with 4 nm steps was performed on all concentrations to assess sample purity.

DNA quantitation in a cuvette or a microplate format was also compared. A 2-fold dilution series from 250 ng/µL to 0.5 ng/µL was transferred to a UVtransparent 96-well microplate at a volume of 200 µL per well or several UV-Vis cuvettes at 1000 µL. Both formats were read on the SpectraMax ABS Plus Microplate reader at 260 nm. A log-log curve fit was applied to each data set, and the standard curves were generated using SoftMax Pro Software.

Protein quantitation

The BCA assay standard curve was prepared by diluting provided BSA according to the assay kit’s protocol. 25 µL of protein standards and 200 µL of the BCA working reagent was transferred to a 96-well clear microplate and incubated for 30 minutes at 37°C. The “BCA” preconfigured protocol was opened in SoftMax Pro Software, and the plate was read at 562 nm using the settings provided. A quadratic curve fit was applied to the data, and a standard curve was generated using SoftMax Pro Software.

Results

The ABS Plus Microplate reader was able to quantitate dsDNA concentration using both low-volume and standard microplate methods. Using the SpectraDrop plate and pre-configured protocol, the ABS Plus microplate reader could detect down to 2 ng/μL using as little as 4 μL of sample, and could automatically measure and calculate relevant parameters such as A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios (Figure 1 and Figure 2). A spectral scan further demonstrated that the samples were contaminant-free (Figure 3).

Figure 1. dsDNA quantitation on the SpectraDrop Micro-Volume Microplate. The ABS Plus reader with the SpectraDrop microplate could detect down to 2 ng/µL and 4 ng/µL of DNA using the 1.0 mm (blue) and 0.5 mm (red) coverslips respectively.

Figure 2. SoftMax Pro Software preconfigured data table. The software’s preconfigured protocols simplify DNA quantitation experiments by automatically calculating absorbance ratios and concentrations of unknown samples.

Figure 3. SoftMax Pro Software preconfigured data table. Spectral scans were performed to assess sample purity. Four representative concentrations of DNA were scanned and only a peak at 260 nm was identified, indicating pure dsDNA.

Alternatively, the ABS Plus microplate reader can quantitate dsDNA in a microplate format. Using a UV-transparent microplate, the reader could measure as little as 0.5 ng/μL dsDNA (Figure 4).

Figure 4. dsDNA quantitation in a 96-well microplate or cuvette. The dsDNA dilution series was detectable from 250 ng/μL to 0.50 ng/μL in both cuvette and microplate formats. Additionally, both dilution series demonstrated high linearity.

Finally, the SpectraMax ABS Plus Microplate Reader can be used to quantitate protein samples with the aid of the BCA assay. Following the provided protocol, BSA standards were measured, and a standard curve was generated comparable to the curve in the assay kit’s literature (Figure 5).

Figure 5. BCA Assay standard curve. The ABS Plus microplate reader was able to detect the BSA standards from the BCA assay. SoftMax Pro Software was used to apply a quadratic curve fit to the data points, and a robust standard curve (r² = 1.000) was generated.

Conclusion

The SpectraMax ABS Plus Microplate Reader is a compact and adaptive microplate reader capable of quantitating nucleic acid and protein samples in various formats such as cuvettes or microplates. When combined with the SpectraDrop Micro-Volume Microplate, the reader can quantitate up to 64 samples with as little as 2 µL sample volume in a single read. Additionally, SoftMax Pro Software’s preconfigured protocols allow for optimized experimentation and faster time to results.

介绍

Hoang Ha | Applications Scientist | Molecular Devices

在遗传学和分子生物学中，对核酸和蛋白进 行定量是许多复杂实验必要的上游检测。虽 然已有多种检测方法，但是最常用的仍然是 紫外分光光度法。紫外分光光度法的原理是 利用分子在固定波长范围吸收光和散射光， 在朗伯比尔定律 ( 方程式 1 ) 的基础上计算待 测物质的浓度。这种方法还需要提前知道样 品的摩尔消光系数和通路长度。

A = εcL

方程式1：朗伯比尔定律是指吸光度 (A) 等于 分子摩尔消光系数 (ε) 乘以浓度 (c) 和通路长 度 (L)。变换等式后我们可以利用吸光度来计 算浓度。

核酸定量已经是一门非常成熟的检测技术 了，并且与最初相比其检测原理并未发生太 多的改变。我们可以在 260 nm 波长下检测 核酸浓度，并且可以利用 230 nm 和 280 nm 波长检测样本纯度。

我们同样可以用紫外分光光度法对蛋白质样 本进行定量，然而除此之外还有更多更准确 的比色测定法。紫外分光光度法利用色氨酸 的芳香结构特性吸收 280 nm 波长的光，浓度 的计算依赖于氨基酸序列上的不同含量的色 氨酸残基。另外，二喹啉甲酸 ( BCA ) 蛋白实 验可以在不依赖于氨基酸序列和长度的情况 下检测蛋白浓度。此方法是在碱性条件下利 用双缩脲反应使氨基酸骨架和铜离子形成带 颜色的螯合物进行测定的。

SpectraMax® ABS Plus 是一款理想的进 行这些定量实验的紫外可见光谱微孔读板 机。在这里，我们展示了几种不同的将 ABS Plus reader 和 SpectraDrop™ MicroVolume Microplate and SoftMax® Pro Software 相结合用来定量检测双链 DNA 和牛血清白蛋白 ( BSA ) 的方法。

材料

• SpectraMax ABS Plus 微孔板读板机 (Molecular Devices cat. #ABS Plus)
• SpectraDrop 超微量板 (Molecular Devices cat. #0200-6262)
• 96 孔平底透明塑料板 (Greiner Bio-One cat. #655101)
• UltraPure™ 小牛胸腺 DNA 溶液 (ThermoFisher Scientific cat. #15633
• Pierce BCA 蛋白质定量试剂盒 (ThermoFisher Scientif
• Pierce™ 牛血清蛋白标准品 Ampules, 2 mg/mL (ThermoFisher Scientific cat. #23209)
• UV-Star® 96 孔板 (Greiner Bio-One cat. #655801)

方法

DNA 定量

1000 ng/µL 的超纯小牛胸腺双链 DNA， 用1x PBS 倍比稀释。将 2 或 4 µL 样品加 入 64 孔 SpectraDrop 微量板中，并用 0.5 mm 或 1.0 mm 盖玻片盖住样品。打 开 SoftMax Pro 软件中预先设置好的程序 “SpectraDrop DNA Quantitation”， 按照默认设置对板进行读数。数据结果用 双对数曲线进行表示，SoftMax Pro 软件 会进一步生成标准曲线。另外，通过范围 为 220 nm 至 350 nm，步进为 4 mm 的 光谱扫描可以检测样品纯度。

我们也比较了运用比色皿和微孔板进行 DNA 定量检测的优劣。待测样品按照倍 比稀释为 250 ng/µL 至 0.5 ng/µL，然后 加入紫外透明 96 孔微孔板中 ( 每孔 200 µL )或者紫外可见比色皿中 (1000 µL)。 我们用 SpectraMax ABS Plus 微孔读板 机在 260 nm 对样本进行了检测。数据结 果用双对数曲线进行表示，SoftMax Pro 软 件会进一步生成标准曲线。

蛋白质定量

按照 BCA 实验试剂盒提供的步骤稀释蛋白 样本并做标准曲线。将 25 µL 蛋白标准品 和 200 µL BCA 反应液加入 96 孔微孔板中 37°C 孵育 30 min。用 SoftMax Pro 软件 打开预设程序“BCA”，在 562 nm 波长 条件下读数。数据用二次方曲线拟合，进 而用 SoftMax Pro 软件生成标准曲线。

结果

ABS Plus 微孔读板机可以在微量和标准微 孔板条件下定量检测双链 DNA 浓度。利用 SpectraDrop 微量板和预设程序，ABS Plus 微孔读板机可以检测到浓度低至 2 ng/µL 体积为 4 µL 的样本，并且可以同时测量 计算相关参数如 A260 / A280 和 A260 / A230 比 例 ( 图 1 和 图 2 )。光谱扫描可以进一步 检测样本是否被污染 ( 图 3 )。另外，ABS Plus 微孔读板机可以用微孔板方法对双链 DNA 定量。利用紫外区透光型微孔板，读 板机可以检测浓度低至 0.02 ng/µL 的双链 DNA ( 图 4 )。最后，SpectraMax ABS Plus 微孔读板机可以在 BCA 浓度测定法 的基础上检测蛋白浓度。按照试剂盒说 明书的步骤检测 BSA 标准品浓度并绘制 标准曲线 ( 图 5 )。

图 1 利用 SpectraDrop 超微量板进行 dsDNA 的定量 ABS Plus 读板机配置 SpectraDrop 微孔板后，对 DNA 的检测灵敏度可以降至 2 ng/μL 和 4 ng/μL，图中所示为检测 DNA 的浓 度曲线，其中蓝色为 1 mm 盖玻片，红色为 0.5 mm 盖玻片

图 2 SoftMax Pro 软件生成数据表 软件预置模板可以自动计算吸光度比值和样品浓度，大大 简化了 DNA 定量实验操作过程

图 3 SoftMax Pro 软件扫描光谱示意图 光谱扫描可以检测样品纯度，四种不同浓度的 DNA扫 描曲线如图所示，都显示只在 260 nm 有峰，说明样品纯度较高

Alternatively, the ABS Plus microplate reader can quantitate dsDNA in a microplate format. Using a UV-transparent microplate, the reader could measure as little as 0.5 ng/μL dsDNA (Figure 4).

图 4 用 96 孔板或比色杯进行 dsDNA 定量 dsDNA 浓度从等比稀释液 250 ng/μL 到 0.50 ng/μL，分 别加入比色杯和微孔板检测，结果显示两种检测方法得到的曲线都具有良好的线性

Finally, the SpectraMax ABS Plus Microplate Reader can be used to quantitate protein samples with the aid of the BCA assay. Following the provided protocol, BSA standards were measured, and a standard curve was generated comparable to the curve in the assay kit’s literature (Figure 5).

图 5 BCA 检测标准曲线 SpectraMax ABS Plus 微孔读板机可以检测 BCA，SoftMax Pro 软件 可利用生成的二次方标准曲线计算出样品浓度，标准曲线 r2 = 1.000

结论

SpectraMax ABS Plus 微孔读板机是一款 简单可靠的仪器，支持多种检测方法，如 比色皿和微孔板，进行核酸和蛋白质定量 的微孔板检测仪器。如果和 SpectraDrop 超微量板搭配使用，此读板机可以一次定 量 64 个体积低至 2 µL 的样本。另外， SoftMax Pro 软件的预设模板程序可以优 化实验条件并且能更快得到结果。

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