Name: protein purification with sec and imac
Uploaded: 2023-04-30T06:30:54.000Z
Duration: 7 min 51 s
Channel: JoVE

K12 Standards

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New York State Science Learning Standards

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Life Science

matter-and-energy-in-organisms-and-ecosystems

Matter and Energy in Organisms and Ecosystems

HS-LS1-6.

chromatography-based-biomolecule-purification-methods

Chromatography-based Biomolecule Purification Methods

在生物化学中，基于色谱的纯化方法被用于从复杂混合物中分离化合物。生物化学家常用的两种方法是凝胶过滤色谱和亲和色谱。在凝胶过滤色谱中，填充有多孔微珠的色谱柱根据分子大小对混合物中的组分进行分离。而亲和色谱则利用含有针对特定目标分子的配体的固定相色谱柱，实现对生物大分子更特异性的分离。
本视频介绍了尺寸排阻色谱和亲和色谱的基本原理及其相关概念。详细描述了通过固定化金属离子亲和色谱纯化组氨酸标签蛋白的逐步操作流程。同时还概述了这两种色谱技术在生物化学与生物医学研究中的应用。

基于色谱的生物分子纯化方法

在生物化学中，基于色谱的纯化方法被用于从复杂混合物中分离化合物。生物化学家常用的两种此类方法是凝胶过滤色谱和亲和色谱。在凝胶过滤色谱中，填充有多孔微珠的色谱柱根据分子大小对混合物中的组分进行分离。而亲和色谱则利用含有针对特定目标分子的配体的固定相色谱柱，实现对生物大分子更特异性的分离。
本视频介绍了尺寸排阻色谱和亲和色谱的基本原理及其相关概念。详细描述了通过固定化金属亲和色谱纯化组氨酸标签蛋白的逐步操作流程。同时概述了这两种色谱技术在生物化学与生物医学研究中的应用。
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&#34;色谱法&#34;指用于从复杂混合物中分离出某一组分的多种方法，这是在确定生物分子的性质和活性之前必不可少的步骤。每种色谱技术根据样品基质和目标化合物的不同，采用不同的分离机制。本视频将重点介绍生物化学中两种常用方法的原理与操作：尺寸排阻色谱和亲和色谱。
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凝胶过滤色谱法或称尺寸排阻色谱法（SEC）是基于样品中化合物的分子大小进行分离的。将含有样品的流动相加入到装有孔状材料（即固定相）的色谱柱中。样品中的分子可分为以下三类。
因体积过大而无法进入孔隙的分子在柱中经过的路径最短。任何分子量超过此&#34;排阻极限&#34;的物质都会同时从柱中流出。而足够小、可自由进入孔隙的分子则会被保留最长时间，并一同流出柱子。允许分子完全进入孔隙的分子量称为&#34;渗透极限&#34;。
只有分子量介于这两个极限之间的分子才会彼此分离，因为它们扩散进入和离开孔隙的时间各不相同。较小的分子在柱上保留时间更长，因为它们在固定相中停留的时间更久；而在此范围内的较大分子则较早流出。
跨越1至2个数量级的分子量通常落在这一分离范围内。因此在选择色谱柱时需考虑这一点，若目标化合物的分子量范围较宽，也可串联使用多个色谱柱。
现在您已经了解了SEC的基本原理，接下来让我们看看该技术的具体操作过程。
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开始凝胶过滤层析（SEC）操作时，需先用去离子水和层析缓冲液对色谱柱进行平衡。准备就绪后，将含有样品的缓冲液注入色谱柱中。随后以低流速推动缓冲液通过色谱柱。检测器监测从色谱柱流出的组分，以确定目标分析物的存在。分子量超过排阻限的大分子会同时从色谱柱中流出。从色谱柱中流出的小分子组分被收集到各收集管中。通过凝胶电泳或其他分析技术，对每一份收集的组分进行检测，以评估目标分子的质量。
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现在让我们来看亲和层析（affinity chromatography，AC），这是一种最高效的蛋白质纯化方法。许多生物分子能够选择性地结合特定配体，亲和层析正是利用这一特性，将针对目标分子的特异性配体固定在固定相上。
当混合物流经层析柱时，目标分子会结合到配体上，其余成分则流过柱子。待混合物完全通过后，可根据特异性选择两种洗脱方法之一来收集目标分子。
生物特异性 洗脱可以说具有 &#34;正常-&#34; 或 &#34;反向角色&#34;在正常作用模式的生物特异性洗脱中，加入一种能与已结合的配体竞争结合目标生物分子的试剂。
在反向角色生物特异性洗脱中，一种试剂与目标物竞争结合已固定的配体。第二种洗脱类型， 非特异性 洗脱，通过改变溶液的pH、离子强度或极性，降低靶标与配体的结合。如果某蛋白质无法与可固定化的配体结合，则可表达带有 &#34;标签&#34;：设计用于结合配体的短肽序列。
一种是固定化金属离子亲和层析， &#34;IMAC&#34; 简而言之，该方法利用固定化的金属配体（如镍或钴）与修饰蛋白上的组氨酸残基结合。通过分子生物学技术，可使目标蛋白携带重复的组氨酸残基，称为多聚组氨酸标签（polyhistidine-tag），其通过组氨酸的咪唑侧链与金属结合。结合后，目标蛋白可通过游离咪唑进行逆向特异性洗脱而被收集，并可用于多种后续应用。&#160;了解了亲和层析的原理后，让我们来看一下实验室中的IMAC操作流程。

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在固定化金属亲和层析（IMAC）中，可将固相直接加入流动相与样品的混合物中，使带组氨酸标签的蛋白质发生结合。然后将此悬浮液倒入层析柱中，未结合的组分随流出液进入废液，而悬浮液则保留在柱内。冲洗悬浮液容器以收集残留的树脂和样品，并将其一并加入层析柱中。
搅拌树脂以确保未结合的组分能够自由流动。加入洗涤缓冲液有助于将其冲洗除去。当所有未结合组分均被去除后，将废液容器更换为用于收集目标蛋白的容器。
加入含有咪唑的缓冲液，混匀并静置，以解离目标分子。咪唑与金属结合，从而取代并释放出带标签的蛋白质。收集游离的蛋白质，并重复咪唑洗脱步骤以确保完全回收。为进一歩纯化样品，可在分析前对样品进行尺寸排阻层析（SEC）。
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在了解了这两种技术的原理和操作步骤之后，让我们来看一下它们在生物化学领域中的一些应用方式。
纯化蛋白质的一个常见原因是研究其在疾病中的作用。囊性纤维化是由囊性纤维化跨膜传导调节蛋白（CFTR）的缺陷引起的。在酵母中表达带有组氨酸标签（his-tag）的该蛋白后，可通过亲和层析和尺寸排阻层析分离出该蛋白，进而研究其功能。
在某些情况下，多聚组氨酸标签的存在可能改变蛋白质的结构，从而影响其功能。另一种常用的标签是麦芽糖结合蛋白（maltose-binding protein, MBP），它能够与层析柱中固定的淀粉结合。随后使用麦芽糖将复合物洗脱下来。接着可通过尺寸排阻层析（SEC）切除并去除MBP标签，从而获得纯净的目标蛋白质。
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您刚刚观看了 JoVE 关于尺寸排阻色谱和亲和色谱的视频。该视频介绍了这两种技术的原理，概述了一般操作步骤，并讲解了它们的一些应用。
感谢观看！
</div>

在生物化学中，基于色谱的纯化方法被用于从复杂混合物中分离化合物。两种常用的方法由……

“色谱法”指用于从复杂混合物中分离出某一组分的各种方法，这是在确定生物分子的性质和活性之前必不可少的步骤。每种色谱技术具有不同的分离机制，具体取决于样品基质和目标化合物。本视频将重点介绍生物化学中两种常用方法——凝胶过滤色谱和亲和色谱的基本原理与操作过程。凝胶过滤色谱法或尺寸排阻色谱法（SEC）基于样品中化合物的大小进行分离。将含有样品的流动相加入到装有多孔材料（即固定相）的色谱柱中。样品中的分子可分为以下3类。过大而无法进入凝胶孔径的分子将沿最短路径通过层析柱。任何分子量超过此“排阻极限”的物质都将在同一时间从层析柱中流出。能够自由进入孔径的小分子将被保留最长时间，并一同从层析柱中洗脱。允许分子完全进入孔径的分子量称为“渗透极限”。只有分子量介于这两个范围之间的分子才会彼此分离，因为它们扩散进入和离开孔隙所需的时间各不相同。较小的分子在柱中保留时间更长，因为它们在固定相中停留的时间更久；而在此范围内较大的分子则会较早流出。跨越1到2个数量级的分子量均在此范围内。应根据此原则选择色谱柱，若目标化合物的分子量范围较宽，可串联使用多根色谱柱。现在你已经了解了尺寸排阻色谱（SEC）的理论，接下来让我们看看它是如何操作的。开始进行凝胶过滤层析（SEC）操作时，必须先用去离子水和层析缓冲液对层析柱进行平衡。准备就绪后，将含有样品的缓冲液注入层析柱中。随后以较低流速推动缓冲液通过层析柱。检测器监测从层析柱流出的组分，以确定目标分析物的存在。分子量超过排阻限的大分子会同时从层析柱中流出。从层析柱中流出的小分子组分被收集于各收集管中。每个组分均通过凝胶电泳或其他分析技术检测目标分子的质量。现在让我们来看一下亲和层析（AC），这是纯化蛋白质最有效的方法之一。许多生物分子能够选择性地结合特定配体，亲和层析正是利用了这一特性，通过将针对目标分子的特异性配体固定在固定相上来实现分离纯化。当混合物流经层析柱时，目标分子会结合到配体上，而其他组分则流过柱子。待混合物完全通过后，可根据特异性选择两种洗脱方法之一来收集目标分子。生物特异性洗脱可分为“正向”或“反向”作用模式。在正向作用的生物特异性洗脱中，加入一种试剂，该试剂与已结合的配体竞争结合目标生物分子。在反向作用的生物特异性洗脱中，一种试剂会与目标分子竞争结合固定的配体。第二种洗脱方式为非特异性洗脱，通过改变溶液的 pH、离子强度或极性来降低目标分子与配体之间的结合力。如果某种蛋白质无法结合可固定的配体，则可对该蛋白质进行表达修饰，使其包含一种“标签”：即一段经基因工程设计的短肽序列，可特异性结合该配体。其中一种方法是固定化金属离子亲和层析，简称 IMAC，该方法利用结合在介质上的金属配体（如镍或钴）与修饰蛋白上的组氨酸残基结合。通过分子生物学技术，可使目标蛋白带有重复的组氨酸残基，称为多聚组氨酸标签（polyhistidine-tag），其通过组氨酸残基上的咪唑侧链与金属离子结合。蛋白结合后，可通过游离咪唑进行特异性洗脱，从而回收目标蛋白，并可用于多种后续实验。现在我们已经了解了亲和层析的基本原理，接下来让我们来看一下在实验室中如何进行 IMAC 操作。在IMAC中，可将固定相直接加入流动相与样品的混合物中，以促进带his标签的蛋白质结合。然后将此悬浊液倒入层析柱中，未结合的组分随流出液进入废液，而悬浊液则保留在柱内。随后用冲洗液洗涤悬浊液容器，以收集残留的树脂和样品，并将其一并加入层析柱中。搅拌树脂以确保未结合的组分能够自由流动。加入洗涤缓冲液有助于将其冲洗掉。当所有未结合的组分均被去除后，更换废液收集容器，改为使用容器收集目标蛋白。加入含有咪唑的缓冲液，混匀并静置，以解离目标分子。咪唑与金属结合，取代并释放出带标签的蛋白质。收集游离的蛋白质，并重复咪唑洗脱步骤，以确保完全回收。为进一步纯化样品，可在分析前对样品进行尺寸排阻色谱（SEC）分离。现在我们已经了解了这两种技术的原理和操作步骤，接下来让我们看看它们在生物化学领域中的一些应用方式。纯化蛋白质的常见原因之一是研究其在疾病中的作用。囊性纤维化是由囊性纤维化跨膜传导调节蛋白（cystic fibrosis transmembrane conductance regulator protein，简称CFTR）的缺陷引起的。在酵母中表达带有his标签的该蛋白后，可通过亲和层析和凝胶过滤层析分离纯化该蛋白，进而研究其功能。在某些情况下，多聚组氨酸标签的存在可能会改变蛋白质的结构，从而影响其功能。另一种常见的标签是麦芽糖结合蛋白（maltose-binding protein, MBP），它能够与柱中固定的淀粉结合。随后使用麦芽糖将复合物洗脱下来。然后可通过凝胶过滤层析（SEC）切除并去除MBP，以获得纯净的目标蛋白质。您刚刚观看了 JoVE 关于尺寸排阻色谱和亲和色谱的视频。该视频介绍了这些技术的原理，概述了一般操作步骤，并讲解了这些技术的一些应用。感谢观看！

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基于色谱的生物大分子分离纯化方法

protein purification with sec and imac

Protein purification with size-exclusion chromatography and affinity chromatography helps isolate biomolecules from complex mixtures. These methods are common in biochemistry because purified samples are needed before a biomolecule’s properties and activity can be studied. The video focuses on the principles behind size-exclusion chromatography, affinity chromatography, and immobilized metal affinity chromatography, or IMAC.

Size-exclusion chromatography, or SEC, separates molecules by size. A sample moves through a column packed with porous beads, which make up the stationary phase. Large molecules that cannot enter the pores travel the shortest path and leave first. Smaller molecules enter the pores, spend more time in the column, and leave later. Molecules between the exclusion limit and the permeation limit can be separated from each other, while molecules above or below those limits exit together.

The SEC procedure begins with equilibration of the column using deionized water and chromatography buffer. Sample-containing buffer is then injected, and the liquid is pushed through at a low flow rate. A detector watches what comes out of the column, and fractions are collected in tubes. These fractions can then be checked for the target molecule by gel electrophoresis or other analytical methods.

Affinity chromatography, or AC, uses the fact that many biomolecules bind selectively to certain ligands. In this method, a target-specific ligand is attached to the stationary phase. As the mixture passes through the column, the target binds to the ligand while the rest flows through. The target can then be released by biospecific elution, which may use a competing agent in normal-role or reverse-role form, or by nonspecific elution, which changes pH, ionic strength, or polarity.

If a protein does not naturally bind to an immobilized ligand, it can be engineered with a tag. A common example is a polyhistidine tag, or his-tag. In immobilized metal ion affinity chromatography, nickel or cobalt on the column binds to histidine residues through the imidazole side chain. The tagged protein is then released with free imidazole by reverse-role specific elution and can be used in downstream applications.

The IMAC procedure begins by mixing the stationary phase with the sample so the his-tagged protein can bind. The slurry is poured into the column, and unbound material drains to waste. The resin is rinsed into the column, stirred, and washed to remove leftover unbound components. After that, a container is placed to collect the target protein, and imidazole buffer is added to release it. The imidazole binds the metal, displaces the tagged protein, and the collected step may be repeated to improve recovery. For further cleanup, SEC can be run before analysis.

These purification methods are useful in biochemical and biomedical research. One example is cystic fibrosis research, where the cystic fibrosis transmembrane conductance regulator protein, or CFTR, is grown with a his-tag in yeast and then isolated with affinity and size-exclusion chromatography. In some cases, a polyhistidine tag can change protein structure and affect function. Another tag used in protein work is maltose-binding protein, or MBP, which binds amylose in a column and is released with maltose. The MBP can then be cleaved and removed with SEC to produce the desired pure protein.

Protein Purification with SEC and IMAC

dialysis: diffusion based separation

Dialysis for Buffer Exchange in Biochemistry

Dialysis is a common biochemistry technique for buffer exchange and desalting. It separates molecules by diffusion through a semipermeable membrane, which lets small molecules pass while holding larger biomolecules inside the sample. The membrane is the key part of the setup. Its pores create a molecular weight cutoff, so a membrane such as a 10 kDa membrane usually keeps molecules larger than 10 kilodaltons inside. This cutoff is not exact, because membranes have a range of pore sizes.

enzyme assays and kinetics

Measuring Enzyme Reaction Rates

Enzyme reaction rates can be measured with assays that track how substrate or product levels change over time. Enzymes are proteins, or sometimes RNAs, that speed up chemical reactions by lowering the activation energy needed to start them. They act on a specific substrate and help form a product that supports life processes. Enzymatic reactions are often described in three steps. First, the substrate binds to the enzyme active site and forms an enzyme-substrate complex. That complex can fall...

maldi-tof mass spectrometry

How MALDI-TOF Reads Biomolecules

MALDI-TOF mass spectrometry reads biomolecules by using a laser, a matrix, and a time-of-flight analyzer. MALDI, or matrix-assisted laser desorption ionization, helps fragile molecules keep their structure while they are moved into the mass analyzer. The analyzer then separates compounds based on size and charge. In MALDI, the sample is mixed into a matrix, usually an organic compound with aromatic and conjugated double bonds. A laser pulse strikes the spot, and the matrix absorbs most of the...

tandem mass spectrometry

Tandem Mass Spectrometry for Biomolecule Analysis

Tandem mass spectrometry is used to isolate a biomolecule and break it into smaller pieces so its makeup can be studied. Because biomolecules often have large, complex structures, it can be hard to identify their molecular composition and sequence from a single measurement. By linking mass spectrometers in series, the technique first selects one ion and then analyzes the fragments it produces. The process starts with an ion source that converts the sample into ions. A mass analyzer then...

protein crystallization

Protein Crystals for Structure Analysis

Protein crystals help scientists study protein structure and function. A crystal is a solid lattice made from purified protein molecules. These crystals are useful in structural biology because they reveal details that methods like mass spectrometry and SDS-PAGE cannot show on their own. Protein crystallization is paired with recombinant protein expression and x-ray diffraction to support this work. The process starts with a large amount of very pure protein. Researchers often make that...

chromatography-based biomolecule purification methods

Protein purification with size-exclusion chromatography and affinity chromatography helps isolate biomolecules from complex mixtures. These methods are common in biochemistry because purified samples are needed before a biomolecule’s properties and activity can be studied. The video focuses on the principles behind size-exclusion chromatography, affinity chromatography, and immobilized metal affinity chromatography, or IMAC. Size-exclusion chromatography, or SEC, separates molecules by size. A...

two-dimensional gel electrophoresis

Protein Mapping with 2D Gel Electrophoresis

Protein mapping with 2D gel electrophoresis separates complex protein mixtures across two steps. The method combines isoelectric focusing, or IEF, with sodium dodecyl sulfate polyacrylamide gel electrophoresis, or SDS-PAGE. Together, these methods can resolve thousands of biomolecules from a single sample by their isoelectric point and molecular weight. The first dimension is IEF. Each protein has an isoelectric point, or pI, which is the pH where its net charge is zero. Proteins are loaded...

metabolic labeling

Tracking Cell Chemistry with Metabolic Labels

Metabolic labeling tracks cell chemistry by using chemical analogs that cells take up and build into biomolecules. These labels help scientists follow biochemical changes and modifications inside cells. The labeled products can then be studied with tools such as SDS-PAGE and NMR to learn more about metabolic pathways. Several labeling strategies are shown here. Isotopic labeling uses analogs that match the natural molecule but contain uncommon isotopes, such as carbon-13 and nitrogen-15 in an...

electrophoretic mobility shift assay (emsa)

electrophoretic-mobility-shift-assay-emsa

Detecting Protein-DNA Binding by EMSA

Electrophoretic mobility shift assay, or EMSA, is a biochemical method used to detect binding between proteins and nucleic acids. It is also called a gel shift assay. The test compares how a labeled nucleic acid moves through a gel before and after it binds a protein. EMSA is used to see whether a specific protein binds to a specific nucleic acid. First, the nucleic acid is labeled to make a probe. The transcript describes radioactive phosphorus-32 as a common label. The probe is then mixed...

photometric protein determination

Measuring Protein Concentration with Absorbance

Measuring protein concentration with absorbance is a key step in many biochemical assays. Photometric methods use the fact that samples with more light-absorbing material transmit less light. Because concentration and absorption are directly related, this makes it possible to measure an unknown sample. The core idea comes from the Beer-Lambert Law. This law states that the concentration of a light-absorbing species is proportional to its absorbance. The video also introduces the Bradford assay...

density gradient ultracentrifugation

Sucrose Gradient Separation in an Ultracentrifuge

Sucrose gradient separation in an ultracentrifuge is a lab method for isolating cell parts and biomolecules. It uses differences in density to make particles sink or rise in a liquid. A high-speed ultracentrifuge speeds up this process so components can be separated without destroying them. The basic idea starts with a suspension, which is particles mixed in a liquid solvent. Particles that are denser than the solvent sediment, while less dense particles float. A rotor inside the...

co-immunoprecipitation and pull-down assays

co-immunoprecipitation-and-pull-down-assays

Protein Interaction Capture Methods in Cells

Co-immunoprecipitation and pull-down assays are laboratory methods used to capture stable protein-protein interactions. Protein-protein interactions help control many biological functions, but many of them have not yet been identified. These assays are related to immunoprecipitation, which isolates a target protein from a mixed sample using an antibody. In co-immunoprecipitation, or CoIP, an antibody binds the target protein, and that protein is already attached to another protein that the...

reconstitution of membrane proteins

Membrane Proteins in Liposome Models

Membrane proteins can be moved into liposome models so their function can be studied outside a cell. Reconstitution means restoring an isolated biomolecule to a working form. For membrane proteins, that usually means placing a purified protein into an artificial lipid membrane. Cell membranes are made mainly of phospholipids and membrane proteins. The phospholipids form a bilayer with hydrophilic phosphate heads facing water and hydrophobic fatty acid tails facing inward. Some membrane...

f&#246;rster resonance energy transfer (fret)

FRET as a Molecular Ruler in Cells

Föster resonance energy transfer, or FRET, is a way to study close-range biochemical interactions using light-emitting molecules. A donor fluorophore can pass energy to an acceptor fluorophore when their emission and absorption spectra overlap. This transfer happens only when the pair is very close together, usually within 10 nm. Because FRET depends on distance, it is often called a molecular or spectroscopic ruler. The strength of the signal is measured as FRET efficiency, which describes...

surface plasmon resonance (spr)

SPR Biosensors for Tracking Binding Kinetics

Surface plasmon resonance, or SPR, is a way to track biomolecular binding on a label-free biosensor. It is used to measure molecular affinity, kinetics, specificity, and concentration. The method works with a thin metal layer on a prism, where changes in reflected light reveal interactions in real time. SPR sensors use a thin layer of a noble metal on the face of a prism. Light is reflected from the prism-metal interface into a photodetector. At one specific angle, called the surface plasmon...

biochemistry

本系列介绍了常用的纯化方法, 如亲和层析, 以及分析方法, 如 MALDI。此外, 视频展示了评估生物相互作用和功能的方法, 如 co-immunoprecipitation 和代谢标记。

Explore how size-exclusion chromatography and IMAC separate proteins by size or binding, and see how tagged proteins are purified for research.

Protein Purification with SEC and IMAC | JoVE K12

Overview

Size-Exclusion Chromatography

Operation of Size-Exclusion Chromatography

Affinity Chromatography

Operation of Affinity Chromatography

Applications

Summary

What is Photosynthesis?

Photosynthesis is a multipart, biochemical process that occurs in plants as well as in some bacteria. It captures carbon dioxide and solar energy to ...

Light as Energy

The energy required to carry out photosynthesis is light&#8212; typically electromagnetic radiation from the sun. The range of all possible wavelengths is ...

Anatomy of Chloroplasts

Green algae and plants, including green stems and unripe fruit, harbor chloroplasts&mdash;the vital organelles where photosynthesis takes place. In ...

Photosystem II

The multi-protein complex photosystem II (PS II) harvests photons and transfers their energy through its bound pigments to its reaction center, and ...

Photosystem I

Although structurally similar to photosystem II (PSII), photosystem I (PSI) is has a different electron supplier and electron acceptor.
Both these ...

The Calvin Cycle

Overview

Oxygenic photosynthesis converts approximately 200 billion tons of carbon dioxide (CO2) annually to organic compounds and produces approximately ...

C4 Pathway and CAM

Most plants use the C3 pathway for carbon fixation. However, some plants, such as sugar cane, corn, and cacti that grow in hot conditions, use alternative ...

the-z-scheme-of-electron-transport-in-photosynthesis

The Z-Scheme of Electron Transport in Photosynthesis

The light reactions of photosynthesis assume a linear flow of electrons from water to NADP+. During this process, light energy drives the splitting of ...

Photosynthesis

Autotrophs

Almost all living organisms on Earth depend on photosynthesis, which is the process that converts sunlight energy into a simple sugar called ...

Plant Pigment Chromatography and Floating Leaf Discs in a Vacuum
ExpandOn the day before the experiment, fill a container with water.
Place the spinach ...

Plant Pigment Chromatography
ExpandNOTE: In this experiment you will separate pigments from spinach leaves using chromatography paper. Individual pigments ...

First Law of Thermodynamics

The First Law of Thermodynamics states that energy cannot be created or destroyed, only transformed. This can be demonstrated within a classic food web ...

Photosystems

Photosystems are multiprotein complexes that form the functional units of photosynthesis in plants, algae, and cyanobacteria. They are found embedded in ...

An Introduction to Cell Metabolism

In cells, critical molecules are either built by joining together individual units like amino acids or nucleotides, or broken down into smaller ...

Reconstitution of Membrane Proteins

Reconstitution is the process of returning an isolated biomolecule to its original form or function. This is particularly useful for studying membrane ...

Use a model to illustrate how photosynthesis transforms light energy into stored chemical energy.

HS-LS1-5.

Macromolecules

Biomolecules
Organisms contain a wide variety of organic molecules with numerous functions which depend on the chemical structures and properties of these ...

Solution Preparation
ExpandCAUTION: It is important to always use care when handling reagents. For example, iodine will stain clothes and skin so wear ...

Identifying Macromolecules with Indicator Solutions
ExpandHYPOTHESES: In this activity, the experimental hypothesis might be that the addition of an ...

Introduction to Carbohydrates

Carbohydrates, proteins, and fats are the primary macronutrients in the human diet. However, carbohydrates are the most favored source of energy in the ...

Carbohydrate Metabolism

Sugars as Energy Storage Molecules

Chemistry of Carbohydrates

Carbohydrates are an essential part of the diet in humans and animals. Grains, fruits, and vegetables are natural sources of carbohydrates that provide ...

Oligosaccharide Assembly

Protein glycosylation starts in the ER lumen and continues in the Golgi apparatus. Glycosyltransferases catalyze the addition of sugar molecules or ...

What are Lipids?

lipid-derived-compounds-in-the-human-body

Lipid-derived Compounds in the Human Body

Fats and lipids are crucial components in the human body. Some lipid-derived compounds, such as fat-soluble vitamins, eicosanoids, lipoproteins, and ...

Fats as Energy Storage Molecules

Structure of Lipids

Lipids include a diverse group of compounds that are largely nonpolar in nature. This is because they are hydrocarbons that include mostly nonpolar ...

Proteins and Amino Acids

Overview

Proteins are chains of amino acids that are connected by peptide bonds and folded into a 3-dimensional structure. The side chains of individual ...

Amino acids

Amino acids are the monomers that comprise proteins. Each amino acid has the same fundamental structure, which consists of a central carbon atom, or the ...

Peptide Bonds

A peptide bond covalently attaches amino acids through a dehydration reaction. One amino acid&#39;s carboxyl group and another amino acid&#39;s amino ...

Nucleic Acids and Nucleotides

Nucleic acids are the most important macromolecules for the continuity of life. They carry the cell&#39;s genetic blueprint and have instructions for its ...

Nucleic Acid Structure

The pentose sugar in DNA is deoxyribose, while in RNA the pentose sugar is ribose. The difference between the sugars is the presence of the hydroxyl group ...

Conditions on Early Earth

Around 4 billion years ago, oceans began to condense on earth while volcanic eruptions released nitrogen, carbon dioxide, methane, ammonia, and hydrogen ...

ATP and Macromolecule Synthesis

Biological macromolecules are organic compounds, predominantly composed of carbon atoms. The carbon atoms are covalently bonded with hydrogen, oxygen, ...

Dietary Connections

In biological systems, most metabolic pathways are interconnected. The cellular respiration processes that convert glucose to ATP&#8212;such as ...

From DNA to Protein

The flow of genetic information in cells from DNA to mRNA to protein is described by the central dogma, which states that genes specify the sequence of ...

Overview of Nitrogen Metabolism

Nitrogen is a very important element for life because it is a major constituent of proteins and nucleic acids. It is a macronutrient, and in nature, it is ...

Compounds Essential to Human Function

The human body is composed of cells that are fundamentally made up of several different molecules. These molecules are essential to carry out all ...

an-introduction-to-molecular-developmental-biology

An Introduction to Molecular Developmental Biology

Molecular signals play a major role in the complex processes occurring during embryonic development. These signals regulate activities such as cell ...

Dehydration Synthesis

Dehydration synthesis (also called a condensation reaction) is the chemical process in which two molecules covalently link together to form a new ...

an-introduction-to-saccharomyces-cerevisiae

An Introduction to Saccharomyces cerevisiae

Saccharomyces cerevisiae (commonly known as baker&rsquo;s yeast) is a single-celled eukaryote that is frequently used in scientific research.  S. ...

An Introduction to Transfection

Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell ...

All living organisms on Earth are directly or indirectly dependent on photosynthesis. It is the only biological process that can capture energy from ...

an-overview-of-alkenone-biomarker-analysis-for-paleothermometry

An Overview of Alkenone Biomarker Analysis for Paleothermometry

Source: Laboratory of Jeff Salacup&#160;- University of Massachusetts Amherst
Throughout this series of videos, natural samples were extracted and ...

an-introduction-to-caenorhabditis-elegans

An Introduction to Caenorhabditis elegans

Caenorhabditis elegans is a microscopic, soil-dwelling roundworm that has been powerfully used as a model organism since the early 1970&rsquo;s.  It was ...

An Overview of Epigenetics

Since the early days of genetics research, scientists have noted certain heritable phenotypic differences that are not due to differences in the ...

An Introduction to Cell Death

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as ...

An Overview of Gene Expression

Gene expression is the complex process where a cell uses its genetic information to make functional products. This process is regulated at multiple ...

An Introduction to Cell Division

Cell division is the process by which a parent cell divides and gives rise to two or more daughter cells. It is a means of reproduction for single-cell ...

An Overview of Genetic Analysis

An organism&#8217;s physical traits, or phenotype, are a product of its genotype, which is the combination of alleles (gene variants) inherited from its ...

an-introduction-to-cell-motility-and-migration

An Introduction to Cell Motility and Migration

Cell motility and migration play important roles in both normal biology and in disease. On one hand, migration allows cells to generate complex tissues ...

An Overview of Genetic Engineering

Genetic engineering &#8211; the process of purposefully altering an organism&#8217;s DNA &#8211; has been used to create powerful research tools and model ...

an-introduction-to-cellular-and-molecular-neuroscience

An Introduction to Cellular and Molecular Neuroscience

Cellular and molecular neuroscience is one of the newest and fastest growing subdisciplines in neuroscience. By investigating the influences of genes, ...

An Overview of Genetics and Disease

Many human diseases are associated with mutations or variations in genetic sequences. Some of these genetic variants are heritable, passed down from ...

an-introduction-to-developmental-genetics

An Introduction to Developmental Genetics

Development is the complex process through which a single-celled embryo transforms into a multicellular organism. Developmental processes are guided by ...

an-overview-of-bgdgt-biomarker-analysis-for-paleoclimatology

An Overview of bGDGT Biomarker Analysis for Paleoclimatology

Annexin V and Propidium Iodide Labeling

Staining with annexin V and propidium iodide (PI) provides researchers with a way to identify different types of cell death&#8212;either necrosis or ...

Co-Immunoprecipitation and Pull-Down Assays

Co-immunoprecipitation (CoIP) and pull-down assays are closely related methods to identify stable protein-protein interactions. These methods are related ...

Construct and revise an explanation based on evidence for how carbon, hydrogen, and oxygen from sugar molecules may combine with other elements such as nitrogen, sulfur, and phosphorus to form amino acids and other carbon-based molecules.

Protein Purification with SEC and IMAC

Description

Transcript

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