Biochemistry Lab: Principles and Key Operations | 生物化学实验：原理与操作要点

📚 Biochemistry Lab: Principles and Key Operations | 生物化学实验：原理与操作要点

Biochemistry experiments require a solid understanding of underlying chemical principles and precise manual skills. This article outlines the core theories and operational highlights for common biochemical assays, from buffer preparation to data analysis.

生物化学实验既需要扎实的化学原理支撑，也需要精准的操作技能。本文围绕常见生化检测技术，从缓冲液配制到数据分析，系统梳理核心理论与操作要点，帮助学习者将实验原理与实际步骤紧密结合。

1. Laboratory Safety and Sample Preparation | 实验室安全与样品准备

Always wear gloves, goggles, and a lab coat when handling biological samples and chemical reagents. All samples should be treated as potentially infectious, and waste must be disposed of according to institutional rules.

接触生物样品和化学试剂时，务必佩戴手套、护目镜和实验服。所有样品都应视为潜在感染源，废弃物须按机构规定处理。

Use a fume hood when working with volatile organic solvents or concentrated acids.

使用挥发性有机溶剂或浓酸时，需在通风橱内操作。
Label all tubes clearly with sample name, date, and concentration.

所有离心管需清晰标注样品名称、日期和浓度。
Prepare samples on ice to prevent enzymatic degradation or microbial growth.

样品应在冰上准备，以防酶降解或微生物滋生。

2. Buffer Solutions and pH Control | 缓冲液与pH控制

A buffer resists pH changes when small amounts of acid or base are added. In biochemistry, phosphate, Tris, and acetate buffers are commonly used to maintain the pH within a narrow range optimal for enzymes or proteins.

缓冲液能在加入少量酸或碱时抵抗pH变化。生物化学中常用磷酸盐、Tris和乙酸盐缓冲液，将pH维持在酶或蛋白质最适的狭小范围内。

Henderson–Hasselbalch equation: pH = pKₐ + log₁₀([A⁻]/[HA])

To prepare a buffer, choose a weak acid whose pKₐ is close to the desired pH. Mix the conjugate acid and base in the ratio predicted by the equation, then verify with a calibrated pH meter.

配制缓冲液时，应选择pKₐ接近目标pH的弱酸。按照方程计算共轭酸和碱的比例混合，再用校准过的pH计验证。

Always calibrate the pH meter with fresh standard buffer solutions.

使用新鲜标准缓冲液校准pH计。
Adjust the volume to the final mark after pH adjustment, because adding HCl or NaOH changes the concentration.

调节pH后再定容至最终刻度，因为加入HCl或NaOH会改变浓度。

3. Spectrophotometry: Principle and Measurement | 分光光度法：原理与测量

Spectrophotometry measures the amount of light absorbed by a solution. The absorbance A is related to concentration c by the Beer–Lambert law.

分光光度法测量溶液对光的吸收量。吸光度A与浓度c的关系遵循比尔–朗伯定律。

A = log₁₀(I₀/I) = ε c l

where ε is the molar absorptivity, c is concentration, and l is the path length (usually 1 cm). The wavelength should be chosen near the absorption maximum of the analyte.

其中ε为摩尔吸光系数，c为浓度，l为光程（通常为1 cm）。所选波长应接近待测物的最大吸收峰。

Zero the spectrophotometer with a blank solution to correct for cuvette and solvent effects.

使用空白溶液对分光光度计调零，以校正比色皿和溶剂的影响。
Hold cuvettes by the frosted sides to avoid fingerprints on the optical faces.

手持比色皿的磨砂面，避免指纹污染透光面。
Ensure the absorbance reading is between 0.1 and 1.0 for reliable linearity.

确保吸光度读数在0.1–1.0之间，以保证线性可靠。

4. Protein Quantification | 蛋白质定量测定

Common methods include the Biuret assay, Bradford assay, and BCA assay. The Bradford dye binds to basic amino acids, shifting its absorption from 465 nm to 595 nm, while BCA relies on the reduction of Cu²⁺ to Cu¹⁺ under alkaline conditions.

常用方法包括双缩脲法、Bradford法和BCA法。Bradford染料结合碱性氨基酸后，最大吸收从465 nm移至595 nm；BCA法依靠碱性条件下Cu²⁺被还原为Cu¹⁺。

Prepare a standard curve using known concentrations of bovine serum albumin (BSA).

使用已知浓度的牛血清白蛋白（BSA）制备标准曲线。
Run samples and standards in triplicate to reduce pipetting error.

样品和标准品均做三复孔，以减少移液误差。
Incubate at room temperature for the exact time specified in the protocol, as colour development is time-dependent.

在室温精确孵育至规定时间，因为显色具有时间依赖性。

5. Centrifugation: Principles and Techniques | 离心技术：原理与操作

Centrifugation uses centrifugal force to separate particles by density and size. The relative centrifugal field (RCF) is expressed in multiples of gravity.

离心利用离心力按密度和大小分离颗粒。相对离心场（RCF）以重力加速度的倍数表示。

RCF = 1.12 × 10⁻⁵ × r × rpm²

where r is the rotor radius in cm and rpm is revolutions per minute. Balance the tubes in opposite positions exactly, and always cap tubes to prevent aerosol contamination.

其中r为转子半径（cm），rpm为转速（转/分）。离心管必须对称平衡放置，并盖紧以防气溶胶污染。

Use a balance with 0.1 g accuracy to equalize tube masses.

用精度0.1 g的天平平衡各管质量。
Choose a rotor type (fixed-angle or swinging-bucket) appropriate for the separation.

根据分离需要选择转子类型（定角或水平吊篮）。

6. Electrophoresis: Separation in an Electric Field | 电泳：电场中的分离

Biomolecules like DNA and proteins carry charge at a given pH. In an electric field, they migrate toward the opposite electrode. Agarose gels separate DNA by size, while SDS-PAGE separates proteins by molecular weight.

DNA和蛋白质等生物大分子在一定pH下带电荷。在电场中，它们向相反电极迁移。琼脂糖凝胶按大小分离DNA，SDS-PAGE按分子量分离蛋白质。

Use a loading buffer containing glycerol to sink the sample into the well and a tracking dye to monitor migration.

上样缓冲液含甘油使样品沉入孔中，并含示踪染料监测迁移。
Apply a voltage appropriate for the gel size; too high voltage heats the gel and distorts bands.

根据凝胶大小施加合适电压；电压过高会使凝胶发热并导致条带变形。
After electrophoresis, stain the gel to visualise the separated molecules, then destain for clear bands.

电泳完成后，染色以显示分离的分子，再脱色以获得清晰条带。

7. Chromatography: Separation by Partition | 层析：分配分离

Chromatography separates components based on their differential interaction between a mobile phase and a stationary phase. Techniques include size-exclusion, ion-exchange, and affinity chromatography.

层析根据组分在流动相和固定相之间不同的相互作用进行分离。常见类型包括体积排阻、离子交换和亲和层析。

Equilibrate the column with the starting buffer before loading the sample.

上样前用起始缓冲液平衡层析柱。
Apply the sample slowly and evenly onto the top of the resin bed.

将样品缓慢、均匀地加到树脂床表面。
Collect fractions in small volumes and monitor by absorbance or activity assay.

小体积收集组分，并用吸光度或活性测定进行监测。

8. Enzyme Kinetics: Measuring Reaction Rates | 酶动力学：测定反应速率

Enzyme kinetics experiments measure the initial velocity v₀ at various substrate concentrations [S]. The Michaelis–Menten equation describes the hyperbolic relationship.

酶动力学实验测定不同底物浓度[S]下的初速度v₀。米氏方程描述了这种双曲线关系。

v₀ = Vmax × [S] / (Kₘ + [S])

To determine Kₘ and Vmax, use a Lineweaver–Burk plot (1/v₀ vs 1/[S]). Ensure that the amount of product formed is measured during the linear phase of the reaction.

为确定Kₘ和Vmax，可使用Lineweaver–Burk双倒数图（1/v₀对1/[S]）。确保在反应线性阶段测定产物生成量。

Keep enzyme concentration constant and much lower than substrate concentration.

保持酶浓度恒定，且远低于底物浓度。
Start the reaction by adding enzyme, and record absorbance at regular intervals immediately.

通过加入酶启动反应，并立即按固定间隔记录吸光度。

9. Quantitative PCR (qPCR): Amplification and Detection | 定量PCR：扩增与检测

Quantitative PCR monitors DNA amplification in real time using fluorescent dyes or probes. The cycle threshold (Ct) is inversely proportional to the initial amount of target DNA.

定量PCR利用荧光染料或探针实时监测DNA扩增。循环阈值（Ct）与初始靶DNA量成反比。

Prepare a master mix to reduce pipetting variation, then add the template DNA.

配制预混液以减少加样误差，再加入模板DNA。
Include no-template controls and a standard curve for absolute quantification.

设置无模板对照，并制备标准曲线用于绝对定量。
Program the thermal cycler with correct denaturation, annealing, and extension temperatures.

在热循环仪上设置正确的变性、退火和延伸温度。

10. Data Analysis, Errors, and Quality Control | 数据分析、误差与质量控制

Accurate results require careful pipetting, proper calibration, and appropriate statistical treatment. Always calculate the mean and standard deviation for replicate measurements, and identify outliers using acceptable tests.

准确结果依赖于精细移液、正确校准和合适的统计学处理。对重复测量应计算均值和标准偏差，并采用可接受的方法剔除异常值。

Use a calibration curve with at least five standard points covering the expected sample range.

标准曲线至少包含5个标准点，覆盖预期样品浓度范围。
Report data with the correct number of significant figures based on the instrument precision.

根据仪器精度报告正确的有效数字位数。
Record any deviations from the protocol and assess their impact on the results.

记录任何与标准流程的偏差，并评估其对结果的影响。

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Biochemistry Lab: Principles and Key Operations | 生物化学实验：原理与操作要点