The Procedure of Electrophoretic Mobility Shift Assay (EMSA)

Electrophoretic Mobility Shift Assay (EMSA) is a technique for studying the interaction between DNA-binding proteins and their associated DNA-binding sequences, which can be used for qualitative and quantitative analysis. This technique was initially used to study DNA-binding proteins and has since been used to investigate the interaction between RNA-binding proteins and specific RNA sequences.

Experimental Preparation

(1) Reasonable experimental design: Design specific probe experimental groups and non-specific probe control groups according to the research purpose, and add specific antibody groups, specific nucleic acid competition groups, etc. if necessary.

(2) Sample preparation: Total protein, nuclear protein, or purified target protein can be selected for sample extraction. Quantify the sample protein and add an equal amount of protein during the experiment.

(3) Probe preparation: Design different probes according to the experimental requirements and add labels. Nucleic acid can be synthesized and labeled manually, and commercial antibodies for known proteins can also be purchased. Nowadays, most laboratories no longer use radioactive labels, and biotin is relatively more commonly used.

Formation of Protein-Probe Complex

(1) Mix the following components in order in a 0.5 mL centrifuge tube:

(2) After 5 minutes on ice, add 1 μL probe (1 μL control probe for the control group).

(3) Incubate at room temperature (20-23°C) in a PCR machine for 30 minutes.

Gel preparation and electrophoresis

(1) Prepare a 6.5% non-denaturing polyacrylamide gel: (Note: adjust the total volume according to the proportion of reagents)

(2) Prepare the gel according to standard procedures.

(3) Pre-electrophorese the gel at 120V in pre-cooled 0.5X TBE buffer for 10 minutes, and wash the sample loading wells after electrophoresis.

(4) Mix the sample with electrophoresis buffer and load it onto the gel.

(5) Place the electrophoresis chamber on ice or at 4°C and run the gel at a constant voltage of 100V until the buffer front has migrated 2-3 cm from the bottom of the gel (approximately 50-60 minutes, adjust the electrophoresis time and voltage according to the actual situation, but do not run the gel for too long).

Transfer

(1) Soak the gel, membrane, filter paper, and fiber pad in pre-chilled 0.5XTBE buffer.

(2) Assemble the "sandwich" in the following order: fiber pad, filter paper, gel, membrane, filter paper, fiber pad. Pay attention to the electrodes and ensure that the gel is placed on the cathode and the membrane on the anode.

(3) Perform the transfer in pre-chilled 0.5XTBE buffer. The transfer apparatus should be placed on ice or in a cold room, and transfer at a constant voltage of 60V for 1 hour. (Adjust the voltage and time according to actual conditions).

Detection

(1) Remove the transferred membrane and place it in a suitable container with buffer for washing. (Avoid membrane drying throughout the detection process).

(2) Remove the washing buffer, add blocking solution, and shake gently. Seal at room temperature for 20 minutes.

(3) Add an appropriate amount of HRP-labeled streptavidin conjugate and incubate at room temperature for 45 minutes with shaking. (Do not add the enzyme-labeled substance directly to the membrane).

(4) Remove the dilution buffer of the enzyme conjugate, wash the membrane three times with washing buffer, and shake gently at room temperature for 10 minutes each time.

(5) Prepare the reaction substrate and evenly add it to the membrane, incubate at room temperature for 5 minutes. (After adding the substrate, gently cover the membrane with a thin film to ensure uniform coverage of the substrate. Be careful not to produce bubbles).

(6) Use a chemiluminescence imaging system to expose and image. (The exposure time can be adjusted according to the detection method).

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Electrophoretic Mobility Shift Assay (EMSA)