EMSA/Gel-Shift Binding Buffer

Item Number

E745592

• Specifications & Purity: 5X
• Stability And Storage: Store at -20℃ for up to 1 year.
• Storage Temp: Store at -20°C
• Shipped In: Ice chest + Ice pads

Product Description

EMSA/Gel-Shift Binding Buffer (5X) is the binding buffer for EMSA/Gel-Shift assay which is generally used to study protein-DNA interactions and to detect the activation level of transcription factors in cells. EMSA/Gel-Shift Binding Buffer (5X) can be used in EMSA for the binding of nuclear proteins or purified transcription factors to specific double-stranded oligonucleotides.This product contains active ingredients such as poly(dI-dC), DTT, glycerol, EDTA, NaCl, MgCl2 and Tris. The concentration of poly(dI-dC) has been optimized to eliminate non-specific bindings without affecting the specific binding between target transcription factor and the labeled probe.This product is not only suitable for isotope-labeled probes, but also for digoxin or biotin-labeled probes.This product is sufficient for 100 binding reactions between protein and probe.

Precautions：

All other reagents related to EMSA are not provided in this product.Please read the entire manual carefully before starting the experiment.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.

Instructions for Use：

1. Set up the following reactions by adding the reagents in sequence. Please refer to Figure 1 for the assay results. Negative Control ReactionNuclease-Free Water7µlEMSA/Gel-Shift Binding Buffer (5X)2µlNuclear Protein or Purified Transcription Factor0µlLabeled Probe1µlTotal Volume10µlCold Probe Competition ReactionNuclease-Free Water4µlEMSA/Gel-Shift Binding Buffer (5X)2µlNuclear Protein or Purified Transcription Factor2µlUnlabeled Probe 1µlLabeled Probe1µlTotal Volume10µl Super-shift ReactionNuclease-Free Water4µlEMSA/Gel-Shift Binding Buffer (5X)2µlNuclear Protein or Purified Transcription Factor2µlTarget Protein Specific Antibody1µlLabeled Probe1µlTotal Volume10µl Sample ReactionNuclease-Free Water5µlEMSA/Gel-Shift Binding Buffer (5X)2µlNuclear Protein or Purified Transcription Factor2µlLabeled Probe1µlTotal Volume10µl Cold Mutation Probe Competition ReactionNuclease-Free Water4µlEMSA/Gel-Shift Binding Buffer (5X)2µlNuclear Protein or Purified Transcription Factor2µlUnlabeled Probe1µlLabeled Probe1µlTotal Volume10µl2. Before adding the labeled probe, the reaction mix should be mixed well and incubated at room temperature (20-25℃) for 10 minutes to eliminate possible non-specific bindings between probes and proteins. Otherwise allow cold probes react preferentially. After adding the labeled probe, mix well and incubate at room temperature (20-25℃) for 20 minutes.3. Add 1μl of EMSA/Gel-Shift Loading Buffer (colorless, 10X) into each reaction, mix well and load the mixture immediately on a gel. Note: Sometimes bromophenol blue can affect protein-DNA interactions. It is recommended to use the colorless EMSA/Gel-Shift Loading Buffer, or that including a very small amount of blue loading buffer to make observation easier.4. Electrophoresis analysis:Figure 1. The typical EMSA result analyzed with ’s EMSA/Gel-Shift Kit . 1. Negative Control Reaction (labeled probe); 2. Sample Reaction (nucleoprotein containing activated target transcription factor + labeled probe); 3. Cold Probe Competition Reaction (nucleoprotein containing activated target transcription factor + labeled probe + unlabeled probe 100 times the amount of labeled probe); 4. Cold Mutant Probe Competition Reaction (nucleoprotein containing activated target transcription factor + labeled probe + unlabeled mutant probe 100 times the amount of labeled probe); 5. Super-shift Reaction (nucleoprotein containing activated target transcription factor + labeled probe + specific antibody of target transcription factor).