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遺伝子発現制御

Gelshift™ Chemiluminescent EMSA

ラジオアイソトープを用いずにタンパク‐DNA結合活性を簡便にスクリーニングするためのEMSA

Gelshift Chemiluminescent EMSA Assay キットは,検証済みの試薬を用いてタンパク質-DNA結合を特定するための,ラジオアイソトープを用いない 簡便なアッセイです。この電気泳動移動度シフト解析(electrophoretic mobility shift assay ; EMSA)では,細胞抽出物または精製因子を,興味のあるコンセンサス結合部位を含むビオチン標識プローブと反応させます。目的のタンパク質が標的DNAに結合した試料は、標識されたDNAバンドの「シフト」が生じ,DNA単独よりも遅く移動します。詳細については,以下の EMSA Method タブをご覧ください。

Gelshift Chemiluminescent EMSA Assayキット

Gelshift Chemiluminescent EMSA Assay キットは,タンパク質‐DNA結合を研究するために必要な全ての試薬が含まれています。また,ビオチン標識および非標識コントロールDNAとコントロール核抽出物も同梱されています。データや詳細については,以下の Gel Shift タブをご覧ください。キットのマニュアルは,Documents タブをご覧ください。

特殊バッファーを用いて独自のゲルシフト / スーパーシフトアッセイを実施

独自のゲルシフト / スーパーシフトアッセイを行いたい方のために,興味のある転写因子に最適な,特性が異なる特殊な結合バッファーを提供しています。これらのバッファーはGelshift Chemiluminescent EMSA Assayキットでは使用されていません。 詳細については,以下の Binding Buffers タブをご覧ください。

 
Name Format Cat No. 価格 (税抜)  
Gelshift™ Chemiluminescent EMSA 100 rxns 37341 ¥86,000 Buy Now
Binding Buffer B-1 240 µl 37480 ¥28,000 Buy Now
Binding Buffer B-2 240 µl 37481 ¥28,000 Buy Now
Binding Buffer B-3 240 µl 37482 ¥28,000 Buy Now
Binding Buffer B-4 240 µl 37483 ¥28,000 Buy Now
Binding Buffer C-1 240 µl 37484 ¥28,000 Buy Now
Binding Buffer C-2 240 µl 37485 ¥28,000 Buy Now
Binding Buffer C-3 240 µl 37486 ¥28,000 Buy Now
Dilution Buffer A 240 µl 37487 ¥28,000 Buy Now
Stabilizing Solution D 360 µl 37488 ¥28,000 Buy Now

Active Motif's Supershift and Gel shift Buffers have been shown to significantly reduce non-specific background. These buffers are available for purchase to save you the time and inconvenience of optimizing buffers for your own experiments.

Binding Buffers B-1 through B-4 are for incubation of the extract:antibody mixture, while Binding Buffers C-1 through C-3 facilitate binding of the labeled probe.

These buffers are not used with the Gelshift Chemiluminescent EMSA.

Buffer Description
Buffer B-1 Higher pH, poly D(I/C)
Buffer B-2 Neutral pH, poly D(I/C)
Buffer B-3 Neutral pH, poly D(I/C) + detergent
Buffer B-4 Higher pH, Herring Sperm DNA
   
Buffer C-1 Higher pH, no blocking DNA
Buffer C-2 Neutral pH, no blocking DNA
Buffer C-3 Neutral pH, no blocking DNA, detergent

Gelshift or Electrophoretic Mobility Shift (EMSA) Info

Electrophoretic mobility shift assays (EMSA), also known as gel shifts, gel retardation assays or mobility assays can be used to study DNA-protein interactions1-3. The principle behind EMSA relies on the fact that DNA-protein complexes migrate slower than DNA alone in a native polyacrylamide or agarose gel. This difference in electrophoretic separation of DNA-protein complexes can be visualized as a "shift" in migration of the labeled DNA band. This enables researches to screen different nuclear extracts or gene promoters for specific transcription factor DNA binding activity.

Better sensitivity

The non-radioactive format of the Gelshift Chemiluminescent EMSA Assay Kit does not sacrifice sensitivity when compared to 32P or digoxigenin methods.

 
Gelshift chemiluminescent EMSA compared with radiolabeled and digoxigenin labeled assay kits

 

Figure 1: Gelshift Chemiluminescent EMSA Kit provides better sensitivity.

HeLa nuclear extract (6.8 µg) was incubated with 20 fmol Oct-1 duplex DNA labeled for use in either the Gelshift Chemiluminescent EMSA kit, a digoxigenin-based EMSA or with 32P using T4 polynucleotide kinase (40,000 cpm/reaction) for use in a radioactive EMSA. The kits were used according to the manufacturer's instructions. The radioactive EMSA was exposed directly to X-ray film using screens.

 
Sensitivity of Gelshift Chemiluminescent EMSA Kit on four different DNA-protein binding complexes

 

Figure 2: Obtain sensitive results for the DNA-protein complex of interest.

The Gelshift Chemiluminescent EMSA Kit was used to obtain sensitive gel shift results for four different DNA-protein complexes. Biotin-labeled target duplexes were incubated with HeLa nuclear extract (Oct-1, AP1 and NFκB) or the provided Control Nuclear Extract from the kit. Unlabeled competitor sequences were used at 200-fold molar excess over labeled DNA. The Control reactions from the Epstein-Barr Nuclear Antigen (EBNA) system were supplemented with 2.5% glycerol and 0.05% NP-40, while the AP1 reactions were supplemented with 10% glycerol. X-ray film exposure time varied from 2 minutes for the EBNA Control, Oct-1 and AP1 to 5 minutes for NFκB.

References

1. Fried, M. and Crothers, D.M. (1981) Nucleic Acids Res. 9:  6505-6525.
2. Revzin, A. (1989) BioTechniques 7:  346-354.
3. Hendrickson, W. (1985) BioTechniques 3:  198-207.

Gelshift Chemiluminescent EMSA Method

The Gelshift Chemiluminescent EMSA Kit provides a non-radioactive method to detect DNA-protein interactions. In this method, cell extracts or purified factor are incubated with a biotin 3' or 5' end-labeled DNA probe containing the consensus binding site of interest. Samples are then resolved by electrophoresis on a native polyacrylamide gel and transferred to a nylon membrane. The biotin end-labeled DNA probe is detected using streptavidin conjugated to horseradish peroxidase (HRP) and a chemiluminescent substrate. Samples in which the protein of interest bound the target DNA will migrate slower than DNA alone resulting in a "shift" of the labeled DNA band.

Example EMSA Binding Reactions

  1. Biotin-labeled DNA = no shift in the absence of cell extract or purified factor
  2. Biotin-labeled DNA + Extract = DNA shift due to the binding of protein to the labled DNA probe
  3. Biotin-labeled DNA + Extract + Excess Unlabeled DNA = no shift as the excess of unlabeled DNA competes for binding of the target protein in the extract; this reaction verifies the specificity of the protein-DNA interaction
 
The Gelshift Electrophoretic Mobility Shift EMSA Kit control transcription factor-DNA binding reactions

 

Gelshift Chemiluminescent EMSA Advantages

  • Non-radioactive assay
  • Better sensitivity than radioactive or digoxigenin methods
  • Fast procedure can be completed in 5 hours
  • Additional reagents supplied to help optimize conditions for your sample system
  • Includes control DNA and extract to help new users understand the methods as they optimize conditions for their sample systems

Contents & Storage

 The Gelshift Chemiluminescent EMSA Kits contain 10X Binding Buffer, Biotin Control DNA, Unlabeled Control DNA, Control Nuclear Extract, Poly d(I-C), 50% Glycerol, 1% NP-40, 1 M KCl, 100 mM MgCl2, 200 mM EDTA pH 8.0 and 5X Loading Buffer, which are all stored at -20°C. Additionally, the kit includes Streptavidin HRP-conjugate, Chemiluminescent Reagent, Reaction Buffer, Blocking Buffer, 4X Wash Buffer and Substrate Equilibration Buffer which should be stored at 4°C.