TransAM® HIF-1

ELISAをベースとしたHIF-1転写因子のDNA結合活性測定

   

TransAM®は、哺乳動物組織や細胞抽出物を用いて転写因子のDNA結合活性を測定できる高感度なELISAベースのアッセイです。TransAM Kit (比色法)はゲルシフトアッセイよりも10倍高感度です。また、TransAMを使用すると、ラジオアイソトープ不要で5時間以内に定量的な結果が得られるうえ、ハイスループット実験に対応します。さらに、40種以上の標的転写因子に対し、それぞれのキットを提供しております(右のリストおよび下記のDocumentsタブより製品マニュアル(14ページ)をご参照ください)。各キットは96 wellのストリッププレートを含み、各wellには転写因子ごとに特異的な二本鎖オリゴヌクレオチドが固相化してあります。核抽出物または全細胞抽出物を加えると、対象の活性型転写因子はオリゴヌクレオチドのコンセンサス配列に結合します。その後、キットに含まれる活性型転写因子に特異的な抗体を用いて定量を行います。詳細は下記のTransAM® Methodタブをクリックしてご確認ください。

TransAM® HIF-1転写因子ELISAキット

TransAM® HIF-1 Kitは、ポジティブコントロール抽出物の他、活性型Hypoxia Inducible Factor 1 alpha (HIF-1a)の実験に必要な構成品一式を提供します。このHIF-1キットはヒトとサルのサンプルにおいてHIF-1aを検出します。下記のHIF-1 Infoタブをクリックするとデータおよび、より詳細な情報をご覧いただけます。また、キットのマニュアルは下記のDocumentsタブからダウンロードいただけます。

 
Name Format Cat No. Price  
TransAM® HIF-1 1 x 96 rxns 47096 ¥140,000 Buy
5 x 96 rxns 47596 ¥580,000 Buy

HIF-1転写因子とは

転写因子Hypoxia-Inducible Factor 1 (HIF-1)は酸素ホメオスタシスにおける重要な制御因子の1つです。HIF-1は低酸素状態(hypoxia)に対する生理的応答を調節し、心臓発作やガン、脳卒中あるいは慢性肺疾患の病態に関連することが知られています。HIF-1はHIF-1αHIF-1βからなるヘテロダイマーのタンパク質です。HIF-1 βが恒常的に発現するのに対し、HIF-1αの発現は酸素濃度が6%以下の時に誘導されます。HIF-1ヘテロダイマーは低酸素応答エレメント(Hypoxia Response Element; HRE)と呼ばれるコンセンサス配列(5’-RCGTG-3’)に結合します。これまでに、血管新生、エネルギー代謝、赤血球造血、細胞増殖と生存率、血管リモデリングおよび血管運動反応に関連するタンパク質をコードする遺伝子など、数十のHIF-1制御遺伝子が同定されています。

 

Transcription Factor ELISA TransAM HIF-1 sensitivity measures HIF-1&alpha activation

図1:HIF-1αの測定

COS-7細胞を0.15 mMのCoCl2投与群と未処置群に分け、それぞれの細胞群からNuclear Extract Kit (Cat. No. 40010)を用いて核を単離しました。HIF-1αはプロテアソームにより分解されることが知られており、その阻害剤であるCoCl2の投与群は未処置群に比べてHIF-1αの存在量が多いと考えられます。実際、これらの核抽出物をTransAM HIF-1 Kitを用いて評価したところ、CoCl2による活性型の増加と、抽出物の量依存性が確認できました。

TransAM®転写因子活性測定ELISAの利点

転写因子研究は歴史的にゲルシフトアッセイやウェスタンブロット、レポータープラスミドのトランスフェクションを用いて行われてきました。しかしいずれの方法も、多大な時間を要してハイスループット実験に不向きなことに加え、半定量的な結果しか得られませんでした。一方、TransAM®アッセイはゲルシフトアッセイに比べて最大で100倍以上も感度が高く、5時間以内で終えることが可能です。また、TransAM®はラジオアイソトープが不要なうえ、Stripwellの採用により1~96サンプルを幅広いスループットでスクリーニング可能です。さらに、レポータープラスミドのトランスフェクションによる発現のばらつきを回避するために行われきた安定発現細胞株の作製からも解放されます。

なぜTransAM®転写因子活性測定ELISAを使うのか?

  • ゲルシフトアッセイよりも最大で100倍高感度
  • ラジオアイソトープが不要で簡便に定量可能
  • 5時間以内に結果を取得
  • 450 nmの分光測定法により簡便に定量解析が可能
  • 96-stripwellにより幅広いスループットに対応

TransAM®の概略

TransAM®はコンセンサス配列に対する転写因子の結合を調べるのに最適な実験手法です。TransAM®キットには、コンセンサス結合部位を含むオリゴヌクレオチドが固定された96-stripwellプレートが含まれています。各ウェルに核抽出物を添加すると任意の転写因子がオリゴヌクレオチドと特異的に結合します。その後、目的の転写因子に結合する一次抗体、HRP標識二次抗体、発色試薬を順次加えて反応させ、比色定量により高感度に検出します(図1)。

Flow chart of the TransAM DNA binding transcription factor ELISA method for measurement of activated transcription factors
図1:TransAMの作業手順

核抽出物に含まれる活性化された転写因子が、stripwellプレートに固定されたオリゴヌクレオチドと結合する。一次抗体、二次抗体および発色試薬を順次反応させて活性化された転写因子を比色定量する。

*TransAM®はEATからライセンスを取得しています。本製品は研究目的用にのみ販売しておりますので、他の目的でご使用の場合には、アクティブ・モティフまでご連絡ください。

構成品と保存条件

TransAM HIF-1 Kitには抗HIF-1α一次抗体、HRP結合二次抗体、野生型および変異HIF-1オリゴヌクレオチド、COS-7核抽出物(ポジティブコントロール)、ジチオスレイトール、プロテアーゼ阻害剤カクテル、ニシン精子DNA、Lysis Buffer、Binding Buffer、10X Washing Buffer、10X Antibody Binding Buffer、Developing Solution 、Stop Solution、HIF-1 96-wellプレート(1枚または5枚)およびプレートシーラー(1枚または5枚)が含まれます。各試薬等の保存条件は-80℃から室温まで様々ですので詳細はマニュアルをご参照ください。すべての試薬等は適切に保管された場合、6か月間安定であることを保証します。

Search our database of customer publications that have used our TransAM® HIF-1 Kit.


 

Can I use your TransAM kit with my species of sample?

All of the TransAM kits will work with human samples. Some have cross-reactivity with mouse or rat. Each TransAM manual contains cross-reactivity information under the Kit Performance and Benefits section.

What is the recommended developing time for the colorimetric TransAM assays?

A recommended development time is given in the technical data sheet (TDS) for each lot. However, this time is only a guideline. It is important to monitor the color development for your particular samples. The TransAM color chart gives some guidelines for colorimetric assays.

TransAM Color Chart

What is the minimum amount of extract (and minimum number of cells) I can use per well?

Each of our specific TransAM kits has a different detection limit, which is indicated in each manual. For example, our TransAM NF-kB kits can detect binding from as little as 0.5 ug nuclear extract. Using a rough estimate yield of 20 ug nuclear extract from 1 million cells from our Nuclear Extract Kit, 0.5 ug nuclear extract can be obtained from approximately 25,000 cells.

How do I determine how much lysate to use per well?

The optimal protein concentration must be empirically determined. We recommend performing a small titration experiment– 5 ug, 10 ug, 20 ug for nuclear extract, more for whole cell extract, in order to ensure that the amount used per well is within detectable levels. You want to get both the treated and control sample signals within the linear range of the assay. See the TransAM color chart.

Can I increase the volume of sample added per well for the TransAM assay?

Yes, you can increase the total volume of sample added and scale up the reaction, but the ratio of Complete Binding Buffer to Complete Lysis Buffer must remain the same. Please note that there may not be enough reagents to perform all 96-rxns if you scale up and do not exceed a total volume of 100 µl. (Complete Binding Buffer plus sample diluted in Complete Lysis Buffer)

Why do I need to read the absorbance at both OD450nm and OD655nm?

The purpose of the reference wavelength (OD655nm) is to correct or normalize any differences in absorbance that are not due to the analyte. Everything being equal, the reference absorbance should not change between samples. But because of factors like variability in detection equipment, wells, etc. the absorbance can vary. Subtracting the reference from the specific reading: OD450nm - OD655nm accounts for these variations. Often the spectrophotometer or the program automatically does this.

Why do I need to run this assay in duplicates?

We recommend at a minimum running the assay in duplicate to get an idea of technical variability. If your assay has a high variability, we recommend doing triplicates. Plot the data with standard error to get a measure of the variability.

Is a standard curve required?

A standard curve may be used to determine the concentration of the target in your samples. However, determining the exact concentration is not required. In fact, a relative comparison between treated and control samples can be more informative than just the absolute concentration.  Another reason to utilize a standard curve is to determine the correlation between protein concentration and the detected signal and ensure that samples fall within the linear range.  With the TransAM kits, this range has already been determined and as long as your signals read below 2.0 OD450, the samples are within the linear range of the microplate reader’s detectability.

How do I know if the standard curve I generated is reasonable?

The R² value of the linear regression line should be 0.94 or higher for the standard curve. To increase this value, perform more replicates and pipet with precision.

What is the difference between the colorimetric and the chemiluminescent versions of the TransAM kits?

Generally speaking, a chemiluminescent ELISA is more sensitive and offers a greater dynamic range as compared to colorimetric ELISAs.

What’s the difference between the TransAM family kits and the TransAM individual kits?

Our TransAM family kits come with two 96-well plates and antibodies against multiple transcription factors within a family. Each antibody is provided in enough abundance to cover one whole 96-well plate, so there is flexibility to assay two or more related antibodies. Our TransAM individual kits each come with one 96-well plate and enough of one antibody to cover that plate.

What is the TransAM NF-kB Flexi kit?

Not all NF-kB sites are the same and our Flexi kits allow users to design and bind their own custom DNA oligonucleotides to the plate. This allows for the study of NF-kB dimers which bind to specific endogenous DNA sequences.

How should I prepare my nuclear extract?

Any of the following are compatible.

  • Active Motif’s Nuclear Extract Kit (Cat. No. 40010/40410)**
  • Preparation of Nuclear Extract protocol in the Appendix of the TransAM manual.
  • Your own nuclear extract protocol, as long as the lysis buffers do not contain SDS. Be sure to resuspend the nuclear pellet in Complete Lysis Buffer.

** Note that some TransAM kits contain Lysis Buffer AM1 while others contain Lysis Buffer AM2. If working with a TransAM kit containing Lysis Buffer AM1, use the Nuclear Extract Kit as written. If working with a TransAM kit containing Lysis Buffer AM2, prepare the Complete Lysis Buffer in the Nuclear Extract Kit using Lysis Buffer AM2 rather than the AM1 that comes in the kit. If you have already prepared your samples using the Lysis Buffer AM1 from the Nuclear Extract Kit, we recommend that you dilute these 1:3 with the Lysis Buffer AM2 that came with your TransAM assay.

The TransAM manual contains instructions for the preparation of nuclear extract from cultured cells. Do you have a protocol for tissue?

The Active Motif Nuclear Extract Kit (Cat. No. 40010/40410) contains a protocol for creating a nuclear extract from tissue. You can also use your own tissue nuclear extract protocol as long as you resuspend the nuclear pellet in the Complete Lysis Buffer recommended in the TransAM kit manual. Avoid using SDS as it can interfere with the assay.

For the nuclear extraction what should I see under the microscope when I monitor cell lysis?

Under a phase contrast microscope intact cells should appear as a dark central region (nucleus) surrounded by a halo of less dense cytoplasm. In lysed cells, the nuclei will appear as dots surrounded by asymmetric debris. See example below:

Cell lysis

Figure: Images under phase contrast microscope of Jurkat cells before (A) and after (B) cell lysis; published with permission from Ziwei Wang, Tsinghua University.

Do you have any recommendations for nuclear extraction from PBMCs?

PBMCs are more difficult to lyse and can be resistant to lysis by the Hypotonic Buffer. Verify cell lysis efficiency using phase-contract microscopy. If cells are not adequately lysed, use a dounce homogenizer with a small clearance pestle (0.025–0.076 mm) to homogenize the sample or draw the sample through a 28-30 gauge needle several times for mechanical shearing. Active Motif’s Dounce Homogenizers for 1 mL capacity (Cat. No. 40401) and 15 mL capacity (Cat. No. 40415) both come with a small and large clearance pestle.

Do you have any recommendations for nuclear extraction from primary cells?

Primary cells can be challenging because they are typically fragile and can easily lyse with just scraping. Also, the number of cells is often limited. Be sure to monitor cell lysis under the microscope to maximize the extraction yield.

What dounce homogenizer should I use for the nuclear prep?

For cells, we recommend a dounce homogenizer with a small pestle clearance (0.025–0.076 mm) to lyse the cells and release the nuclei. For tissue we recommend a dounce homogenizer with a large pestle clearance (0.089–0.14 mm) to first homogenize the tissue into a single cell slurry and then a dounce homogenizer with a small pestle clearance to lyse the cells and release the nuclei. Active Motif’s Dounce Homogenizers for 1 mL capacity (Cat. No. 40401) and 15 mL capacity (Cat. No. 40415) both come with a small and large clearance pestle.

Clearance pestle

How many times can I freeze-thaw my nuclear extract?

It’s best to aliquot the extracts so that freeze-thaw cycles are not required. However, if you must thaw, we do not recommend freeze-thawing more than 2 times. Multiple freeze-thaws degrade proteins and destroy protein activity. Thaw the extracts on ice using pipetting to mix. Do you not vortex protein samples vigorously.

Can I use frozen samples to create the nuclear extracts?

We recommend using fresh cells or tissue, as the freezing process can disrupt the cytoplasmic and nuclear compartments, making it difficult to get pure fractions. However, if necessary, snap freeze tissues at -80°C. Cryopreserve cells before freezing at -80°C.

I am preparing a nuclear extract. The nuclear pellet is viscous and is not resuspending well in the Complete Lysis Buffer. How can I get it into solution?

To enhance the solubility of the pellet:

  • Pipet up and down a few times with a large bore pipette tip.
  • Use a dounce homogenizer.
  • Vortex on high for 10 seconds.
  • Increase the 30-minute incubation on the rocking platform to an hour.

If needed add 5-10 ul of Complete Lysis Buffer. Be sure not to overdilute the nuclear sample. Note that certain cell types are more likely to produce a viscous nuclear pellet and yet still give good protein yields, even if it doesn’t completely disappear. Do not add detergent as it could interfere with the TransAM assay.

Can I use whole cell extract?

It depends. With a whole-cell extract, the transcription factor of interest will be diluted into a larger volume than in a nuclear extract. So, more protein (2x or more) will be required. Also, if the transcription factor is regulated by nuclear-cytoplasmic localization, using whole-cell extract could affect the functional aspect of the assay.

Can I use the TransAM kit with cytoplasmic extracts?

It is not recommended for several reasons. The TransAM kit is designed to measure protein-DNA binding, which exclusively occurs in the nucleus. Any signal with the cytoplasmic extract may not be physiologically relevant. Also, the cytoplasmic extract is going to be much more dilute as compared to nuclear extract and will contain detergents unlike the nuclear fraction that that may interfere with the assay.

How should I quantify my nuclear extract protein?

We recommend using a Bradford Assay to quantify the protein rather than BCA. A BCA assay requires a higher dilution factor to eliminate confounding interfering agents and can limit the detection of protein in all but very concentrated samples. If using the Nuclear Extract Kit (Cat. No. 40010/40410), we recommend a dilution factor of 1:50 in water. There is a protocol for creating a BSA standard curve in the Appendix of the Nuclear Extract Kit manual.

How many cells/tissue should I use for the nuclear extract?

We suggest isolating nuclear extracts from samples of approximately 8.8 x106 cells or 45 mg tissue. A tissue conversion factor for general estimation is 2x105 cells/mg.

What protein yield can I expect from 8.8 x 106 cells?

This will depend on your cell or tissue type, but in general you should expect 150-250 µg at ~3-5 mg/ml from 8.8 x 106 cells.

What components can interfere with the TransAM assays?

SDS or sodium azide in your cell lysis buffer will inhibit the binding and can render your sample incompatible.