Name: detecting protein-dna binding by emsa
Uploaded: 2023-04-30T06:31:38.000Z
Duration: 6 min 57 s
Channel: JoVE

K12 Standards

new-york-state-science-learning-standards

New York State Science Learning Standards

High School

Life Science

Structure and Function

HS-LS1-1.

electrophoretic-mobility-shift-assay-emsa

Electrophoretic Mobility Shift Assay (EMSA)

ゲルシフトアッセイ (emsa)

ゲルシフトアッセイ (EMSA) は、タンパク質や核酸の間のバインディングを明らかにするために使用生化学的なプロシージャです。このアッセイ標識核酸およびテスト蛋白質が混在します。バインディングは、質量に基づくコンポーネントを分離するゲルの電気泳動によって決められてタメて構造。
このビデオは、EMSA とゲルおよび蛋白質の準備、バインド、電気泳動、検出などの一般的な手順の概念を示しています。このビデオで説明したアプリケーションには、クロマチンの酵素、biontinylation が組み込まれた変更された EMSA の分析と結合性細菌の応答調節物質に関する研究が含まれます。
<div class="chapter_text" id="script_0">
EMSA、電気泳動の移動性シフト試金のゲル シフト試金とも呼ばれますは、汎用性と敏感な生化学的なプロシージャです。EMSA はゲルの電気泳動のバンドの変化に着目したタンパク質と核酸との間のバインディングを解明します。
このビデオは、EMSA の原則を説明、一般的な手順について説明します、いくつかのアプリケーションについて説明します。
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<div class="chapter_text" id="script_1">
DNA 複製、転写と修理、として RNA プロセシングは、すべての重要な生化学的なプロセスです。皆はタンパク質と核酸との結合を含みます。多くの深刻な病気や疾患は、このバインディングの変更に関連付けられます。EMSA は、質的特定の蛋白質が特定の核酸にバインドするかどうかを決定するための手法です。まず、核酸が付いて、通常放射性リン-32、プローブを作成します。その後、テスト蛋白質および核酸プローブを混合します。核酸プローブに結合するタンパク質、結果として得られる複合体は核酸だけよりも異なる構造と質量が大きい。
バインドされた後、複合体はゲル電気泳動で分析されます。この手法では、電界は、高分子ゲルのマトリックスを通して移行するを強制します。コンポーネントは、に基づいて質量・電荷・ コンフォメーションを区切ります。電気泳動は、バインドされていないプローブからタンパク質・ DNA 錯体を区切ることができます。彼らは異なる質量と立体配座を持っている、ので、彼らは異なるレートでゲルを通る移行し、分離します。分離は、放射性リンの存在のおかげで簡単に検出し、蛋白質は特定の核酸に正常にバインドを証明します。蛋白質の識別を確認するには、「supershift 分析」は知られている蛋白質に親和性抗体を使用します。さらに解像度は高く、複合体をシフトの付加的な利点があります。
今、我々 は原則を見てきました、ラボでしてみましょう。
</div>
<div class="chapter_text" id="script_2">
手順を開始するには、タンパク質が分離である必要があります。これを行うには、分子生物学的手法、細胞の蛋白質を表現し、浄化されます。
核酸増幅は、プローブを作成するラベルします。ラベリングは、放射性リン 32 を含む dCTP を 10 分間インキュベーションを介して行われます。放射線の安全なワークベンチと保護具が必要です。
ゲルは、用意しています。ゲル非変化、蛋白質の構造を変更して可能性のある電気泳動中にプローブからバインド解除を防ぐする必要があります。ポリアクリルアミドゲルの長さに 5 を 20 nm と短いプローブ 100 塩基対までの役に立つの細孔径があります。Agarose のゲルは 70-700 nm の細孔径を持ち、大きいプローブ用に便利です。
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<div class="chapter_text" id="script_3">
タンパク質と核酸プローブの準備、我々 はバインドに進みます。トリス緩衝液を用意し、タンパク質とプローブが追加されます。PH を生理的条件と塩濃度のタンパク質が非標的核酸と弱い結合を形成することを防ぐために十分にする必要があります。4 ° C で 20-30 分のため反応が進む
次のステップは電気泳動です。低イオン強度および結合の反作用で使用されるような pH のバッファーが利用されています。錯体を安定化、モビリティを向上させ、熱発生は少なく、「ケージ効果」が得られます。電気泳動後のゲルのコンポーネントは、フィルター紙の上に転送されます。暗い部屋では、フィルター ペーパー、フィルムに公開されます。プローブに結合するタンパク質と、2 つの異なるラベルの付いた領域は転送で表示されます。コンプレックスを表す 1 つと非連結のプローブを表す別の 1 つ。分離は、核酸に蛋白質が正常にバインドされることを示します。
</div>
<div class="chapter_text" id="script_4">
今では基本的な手順を見てきた、いくつかのアプリケーションを見てみましょう。
クロマチンは、DNA および真核細胞に存在する蛋白質の堅く詰められた複合体であります。酵素のクロマチン転写する DNA を開く構造に変更します。複合体の移動を変更、EMSA を使用して酵素の結合活性を探索できます。
分類に別の方法でメチル基転移酵素酵素と DNA との相互作用を活用しています。補足因子は、DNA メチル基転移酵素を介して完全にバインドする変更できます。リン 32 のラベルではなく補因子、ビオチンには放射性ではないために、有利な活用します。補因子は特定の添付ファイルで、遺伝子型、メチル化検出及び遺伝子デリバリーに関連します。Biotinilated 核酸は紫外線蛍光を介して検出されます。
環境の刺激は、ヒスチジンのキナーゼをアクティブにしたとき、「応答レギュレータ」でリン酸化されます。DNA の転写、EMSA によって学ぶことができますに影響を与えるにバインドされ、順番。例えば、興味の遺伝子にバインドするデスルフォビブリオ属尋常性の応答調節因子を示した。EMSA のバインディングが行われたことを確認する使用されました。
</div>
<div class="chapter_text" id="script_5">
ゼウスの電気泳動の移動性シフト試金のビデオを見てきただけ。今操作、その手順とその主要な動作パラメーターの手順の原則を理解する必要があります。
見てくれてありがとう!
</div>

ゲルシフトアッセイ (EMSA)

The electrophoretic mobility shift assay (EMSA) is a biochemical procedure used to elucidate binding between proteins and nucleic acids. In this assay a ...

EMSA(電気泳動移動度シフトアッセイ)は、ゲルシフトアッセイとも呼ばれ、汎用性が高く感度の高い生化学的手順です。EMSAは、ゲル電気泳動におけるバンドのシフトを検出することにより、タンパク質と核酸の間の結合を解明します。このビデオでは、EMSA の原理を説明し、一般的な手順を説明し、いくつかのアプリケーションについて説明します。DNAの複製、転写、修復、およびRNAプロセシングはすべて重要な生化学的プロセスです。それらはすべて、タンパク質と核酸との間の結合を伴います。多くの重篤な疾患や障害は、この結合の修飾に関連しています。EMSAは、特定のタンパク質が特定の核酸に結合するかどうかを定性的に判断する手法です。まず、核酸を通常は放射性リン32で標識してプローブを作成します。次に、試験タンパク質と核酸プローブを混合します。タンパク質が核酸プローブに結合すると、結果として生じる複合体は、核酸単独の場合よりも質量が大きく、コンフォメーションが異なります。結合すると、錯体はゲル電気泳動で分析されます。この手法では、電場によって高分子がゲルマトリックス内を移動するように強制されます。成分は、質量、電荷、およびコンフォメーションに基づいて分離します。電気泳動は、結合していないプローブからタンパク質-DNA複合体を分離することができます。それらは異なる質量とコンフォメーションを持っているため、ゲル内を異なる速度で移動し、分離します。放射性リンの存在により、分離は容易に検出され、タンパク質が所定の核酸に首尾よく結合することが証明されています。タンパク質の同定を確認するために、「スーパーシフトアッセイ」では、タンパク質に対する既知の親和性を持つ抗体を使用します。これには、コンプレックスをさらにシフトし、解像度を向上させるという追加の利点があります。原理を見てきたところで、研究室で見てみましょう。手順を開始するには、タンパク質を単離する必要があります。これを行うために、分子生物学的手法を使用して細胞内でタンパク質を発現させ、その後精製します。核酸を増幅して標識し、プローブを作成します。標識は、放射性リン-32を含むdCTPと10分間インキュベートすることによって行われます。放射線に安全な作業台と保護具が必要です。次に、ゲルを調製します。ゲルは、電気泳動中にタンパク質がコンフォメーションを変化させ、プローブから結合を剥がすのを防ぐために、非変性である必要があります。ポリアクリルアミドゲルの細孔径は5〜20 nmで、長さが最大100塩基対の短いプローブに役立ちます。アガロースゲルの細孔径は70-700 nmで、より大きなプローブに有用です。タンパク質と核酸プローブの準備ができたので、結合に進みます。TRIS緩衝液を調製し、タンパク質とプローブを添加します。pHは生理学的条件と同程度で、タンパク質が非標的核酸と弱い結合を形成するのを防ぐのに十分な塩濃度である必要があります。反応は4°Cで20〜30分間進行する。次のステップは電気泳動です。低イオン強度のバッファーと、結合反応で使用されるものと同様のpHが利用されます。複合体を安定させ、移動性を高め、発熱を抑える「ケージ効果」をもたらします。電気泳動後、ゲルの成分を濾紙に移します。暗い部屋では、濾紙をフィルムにさらします。タンパク質がプローブに結合すると、転写中に2つの異なる標識領域が見えます。1 つは複合施設を表し、もう 1 つは非バインド プローブを表します。この分離は、タンパク質が核酸に正常に結合したことを示しています。基本的な手順を確認したので、いくつかのアプリケーションを見てみましょう。クロマチンは、真核細胞に見られるDNAとタンパク質の密集した複合体です。クロマチンリモデリング酵素は、DNAを転写するために構造を修飾します。これにより複合体の移動度が変化するため、EMSAを使用して酵素の結合活性を調べることができます。標識の別のアプローチは、DNAとメチルトランスフェラーゼ酵素との間の相互作用を利用することです。補因子は、メチルトランスフェラーゼを介してDNAに永久に結合するように修飾することができます。リン32で標識するのではなく、補因子はビオチンに結合しており、放射性ではないため有利です。補因子はその結合において部位特異的であるため、ジェノタイピング、メチル化検出、および遺伝子導入に関連しています。ビオチン化核酸は、紫外線蛍光によって検出されます。環境刺激がヒスチジンキナーゼを活性化すると、「応答調節因子」がリン酸化されます。これは次にDNAに結合し、転写に影響を与え、EMSAで研究することができます。たとえば、レスポンスレギュレーターは?Desulfovibrio vulgarisは、目的の遺伝子に結合することが示されました。EMSA を使用して、バインディングが行われたことを確認しました。JoVEの電気泳動移動度シフトアッセイに関するビデオをご覧になりました。これで、その動作原理、手順のステップ、および主要な動作パラメータを理解できます。ご覧いただきありがとうございます!

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Biochemistry

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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 with the test protein, and any binding changes the size and shape of the complex.

The bound and unbound molecules are then separated by gel electrophoresis. An electric field pulls macromolecules through a gel matrix. They separate by mass, charge, and conformation. Protein-DNA complexes move differently from free probe, so the shifted band shows that binding occurred. A supershift assay can also confirm the protein’s identity. In that step, an antibody with known affinity binds the protein and shifts the complex even further.

The lab procedure begins by isolating the protein. Molecular biology methods are used to express the protein in cells and then purify it. The nucleic acid is amplified and labeled to form the probe. Because phosphorus-32 is radioactive, a radiation-safe workbench and protective equipment are required during labeling.

The gel must be non-denaturing so the protein does not change conformation or fall off the probe during electrophoresis. Polyacrylamide gels have pore sizes of about 5 to 20 nm and are used for short probes up to 100 base pairs long. Agarose gels have larger pores, about 70 to 700 nm, and are used for larger probes. After the protein and probe are ready, they are mixed in TRIS buffer for binding.

The binding reaction uses a pH close to physiological conditions. The salt concentration must be high enough to reduce weak, non-target binding. The reaction runs for 20 to 30 min at 4 °C. Electrophoresis is then done in a buffer with low ionic strength and a similar pH. This creates a caging effect that helps stabilize complexes, increases mobility, and reduces heat generation.

After the gel runs, the components are transferred onto filter paper. The paper is exposed to film in a dark room. If binding occurred, two labeled regions appear: one for the protein-nucleic acid complex and one for the unbound probe. That separation shows the protein successfully bound the nucleic acid.

EMSA is useful in several research areas. Chromatin-remodeling enzymes can be studied because they change chromatin structure and affect DNA mobility. A modified EMSA can use biotinylated nucleic acids instead of radioactive labels. This approach is site-specific and useful for genotyping, methylation detection, and gene delivery. It is also detected through ultraviolet fluorescence.

The assay can also help study bacterial response regulators. When environmental stimuli activate histidine kinases, a response regulator becomes phosphorylated and binds DNA to affect transcription. The transcript gives an example from Desulfovibrio vulgaris, where EMSA was used to confirm binding to a gene of interest. EMSA therefore provides a sensitive way to study protein-nucleic acid interactions and their role in DNA replication, transcription, repair, and RNA processing.

Detecting Protein-DNA Binding by EMSA

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.

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maldi-tof mass spectrometry

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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

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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...

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chromatography-based biomolecule purification methods

chromatography-based-biomolecule-purification-methods

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...

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...

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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, 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

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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

このコレクションでは、親和性クロマトグラフィーなどの一般的に使用される精製方法や、MALDI-TOFなどの分析方法について紹介します。さらに、共同免疫沈降法や代謝標識法など、生体分子の相互作用や機能を評価する方法も紹介します。

Learn how EMSA detects protein-DNA binding with labeled probes, gel electrophoresis, and band shifts. See the steps and common applications.

Detecting Protein-DNA Binding by EMSA | JoVE K12

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

Matter and Energy in Organisms and Ecosystems

HS-LS1-6.

Inheritance and Variation of Traits

HS-LS3-1.

Earth and Space Sciences

Human Sustainability

HS-ESS3-3.

Overview

Principles of EMSA

Protein and Gel Preparation

Binding, Electrophoresis, and Detection

Applications

Summary

Characteristics of Life

Biology is a natural science that studies life and living organisms, including their structure, function, development, interactions, evolution, ...

https://cloudfront.jove.com/files/media/science-education/science-education-thumbs/10647.jpg

Cell Structure

Background
Cells represent the most basic biological units of all organisms, whether it be simple, single-celled organisms like bacteria, or large, ...

Visualizing Onion and Cheek Cells
ExpandImmediately before the experiment, wash and peel onion bulbs for the class.
Remove the entire brown outer skin and ...

Visualizing Onion and Human Cells
ExpandIn this experiment you will prepare different types of cells from a plant and animal, onion and human ...

What are Cells?

Cell Diversity

The concept of a cell started with microscopic observations of dead cork tissue by Robert Hooke in 1665. Hooke coined the term &#34;cell&#34; based on the ...

Cytoplasm

The cytoplasm consists of organelles and a framework of protein scaffolds called the cytoskeleton suspended in an aqueous solution, the cytosol. The ...

The Nucleus

The nucleus is a membrane-bound organelle that acts as a control center in a eukaryotic cell. It contains chromosomal DNA, which controls gene expression ...

The Central Dogma

The DNA Helix

Nucleic Acids

DNA as a Genetic Template

Structure of a Gene

A gene is the fundamental unit of heredity. Every individual has two copies of each gene, one inherited from each parent. Although most people contain the ...

Chromosome Structure

Base-pairing and DNA Repair

What is Gene Expression?

A gene is a stretch of DNA that serves as the blueprint for functional RNAs and proteins. Since DNA is comprised&#160; of nucleotides and proteins are ...

RNA Structure

The basic structure of RNA consists of a string of ribonucleotides attached by phosphodiester bonds. Although most RNA is single-stranded, it can form ...

Types of RNA

Three main types of RNA are involved in protein synthesis: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). These RNAs perform diverse ...

Transcription

Transcription is the synthesis of RNA from a DNA sequence by RNA polymerase. It is the first step in producing a protein from a gene sequence. ...

Translation

Translation is the process of synthesizing proteins from the genetic information carried by messenger RNA (mRNA). Following transcription, it constitutes ...

Ribosomes

Ribosomes translate genetic information encoded by messenger RNA (mRNA) into proteins. Both prokaryotic and eukaryotic cells have ribosomes. Cells that ...

Protein and Protein Structures

Proteins: From Genes to Degradation

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 ...

Protein Folding

Proteins are chains of amino acids linked together by peptide bonds. Upon synthesis, a protein folds into a three-dimensional conformation, critical to ...

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 ...

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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 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 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 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 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 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-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, ...

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 ...

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 ...

Bacterial Transformation: Electroporation

The term &#x201C;transformation&#x201D; refers cellular ingestion of foreign DNA.  In nature, transformation can occur in certain types of bacteria.  In ...

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 ...

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Bacterial Transformation: The Heat Shock Method

Transformation is the process that occurs when a cell ingests foreign DNA from its surroundings. Transformation can occur in nature in certain types of ...

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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. ...

Cell Cycle Analysis

Cell cycle refers to the set of events through which a cell grows, replicates its genome, and ultimately divides into two daughter cells through the ...

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 ...

Chromatin Immunoprecipitation

Histones are proteins that help organize DNA in eukaryotic nuclei by serving as &#8220;scaffolds&#8221; around which DNA can be wrapped, forming a complex ...

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 ...

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Community DNA Extraction from Bacterial Colonies

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Luisa Ikner
Traditional methods of analysis ...

Cytogenetics

Cytogenetics is the field of study devoted to chromosomes, and involves the direct observation of a cell&#8217;s chromosomal number and structure, ...

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Embryonic Stem Cell Culture and Differentiation

Culturing embryonic stem (ES) cells requires conditions that maintain these cells in an undifferentiated state to preserve their capacity for self-renewal ...

DNA Gel Electrophoresis

DNA gel electrophoresis is a technique used for the detection and separation of DNA molecules. An electric field is applied to a gel matrix comprised of ...

Enzyme Assays and Kinetics

Enzyme kinetics describes the catalytic effects of enzymes, which are biomolecules that facilitate chemical reactions necessary for living organisms. ...

DNA Ligation Reactions

In molecular biology, ligation refers to the joining of two DNA fragments through the formation of a phosphodiester bond.  An enzyme known as a ligase ...

explant-culture-for-developmental-studies

Explant Culture for Developmental Studies

Explant culture is a technique in which living cells or tissues are removed from an embryo for continued development outside of the organism. This ex vivo ...

DNA Methylation Analysis

Methylation at CpG dinucleotides is a chemical modification of DNA hypothesized to play important roles in regulating gene expression. In particular, the ...

Expression Profiling with Microarrays

Microarrays are important tools for profiling gene expression, and are based on complementary binding between probes that are attached to glass chips and ...

Density Gradient Ultracentrifugation

Density gradient ultracentrifugation is a common technique used to isolate and purify biomolecules and cell structures. This technique exploits the fact ...

F&#246;rster Resonance Energy Transfer (FRET)

F&#246;rster resonance energy transfer (FRET) is a phenomenon used to investigate close-range biochemical interactions. In FRET, a donor photoluminescent ...

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Detecting Environmental Microorganisms with the Polymerase Chain Reaction and Gel Electrophoresis

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Bradley Schmitz
Polymerase chain reaction (PCR) is ...

Gel Purification

Gel purification is used to recover DNA fragments after electrophoretic separation. DNA recovery from an agarose gel includes three basic steps: binding, ...

Detecting Reactive Oxygen Species

Reactive oxygen species are chemically active, oxygen-derived molecules capable of oxidizing other molecules. Because of their reactive nature, there are ...

Construct an explanation based on evidence for how the structure of DNA determines the structure of proteins which carry out the essential functions of life through systems of specialized cells.

Detecting Protein-DNA Binding by EMSA

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