Your Step-by-Step Guide to Electrophoretic Mobility Shift Assay

The Electrophoretic Mobility Shift Assay (EMSA), or gel shift assay is a basic and rapid method to detect protein complex with nucleic acids. Originally utilized broadly in the investigation of sequence-specific DNA-binding proteins such as transcription factors, EMSA has been additionally developed to explore DNA-protein interactions, RNA-protein interactions. It is likewise connected to qualify an quantify proteins that particularly tie to given nucleotides, empowering to accommodate a wide range of binding conditions.

Principles

The purified protein and the cell crude extract are usually incubated with the 32P isotope-labeled DNA or RNA probe, and the complex and the unbound probe are isolated on the non-denaturing polypropylene gel electrophoresis. DNA-complexes or RNA-complexes move slower than non-bound probes. Isotope-labeled probes differ depending on the binding protein studied, double-stranded or single-stranded can both be ok. When detecting DNA binding proteins such as transcriptional regulatory factors, purified proteins, partially purified proteins, or nuclear cell extracts can be used. In the detection of RNA binding protein, according to the location of the target RNA binding protein, purified or partially purified protein, as well as nuclear or cytoplasmic cell extract can also be used . The DNA or RNA fragments and oligonucleotide fragments (specific) containing protein binding sequences, and other non-relevant fragments (non-specific), were used in competitive experiments to determine the specificity of DNA or RNA-binding proteins. Specific binding is determined according to the characteristics and intensity of the complex in the presence of competing specific and non-specific fragments.

Experimental Materials

DNA samples

Reagents, kits

[γ-32P]ATP, T4 polynucleotide kinase, Nuclease-Free Water, T4 polynucleotide kinase buffer, Ammonium acetate, TE, Anhydrous ethanol, TBE buffer, steaming water, Methylene bisacrylamide, Acrylamide, glycerin, Ammonium persulfate, TEMED (tetramethylethylenediamine), EMSA Gel-Shift binds buffer, BPB

Equipment

Water bath, PCR, Centrifuge, Electrophoresis, Electrophoresis tank

Operating Method

(1) Probe labeling

1. Set the probe reaction system as follows:

a. Probe to be labeled (1.75 pmol / μl): 2 μl

b. T4 Polynucleotide Kinase Buffer (10X): 1 μl

c. Nuclease-Free Water: 5 μl

d. [γ-32P] ATP (3 000 Ci / mmol at 10 mCi / ml): 1 μl

e. T4 Polynucleotide Kinase (5–10 u / μl): 1 μl

f. the total volume to be 10 microliters

g. followed by adding a variety of reagents, add isotopes, Vortex mix, then add T4 Polynucleotide Kinase, and mix.

2. Use a water bath or PCR instrument at 37 ° C for 10 minutes.

3. Add 1 μl of the probe to mark the stop solution, mix and terminate the probe labeling reaction.

4. Add 89 μl of TE to mix. At this point you can take a small amount of probe for the detection of the efficiency of the labeling. Usually it’s more than 30%, that is, more than 30% of total radioactivity labeled to the probe. For the sake of simplicity of the experiment, it is not necessary to determine the labeling efficiency of the probe.

5. The labeled probe should better be used immediately, or no longer than 3 days. The labeled probe can be stored at -20 ° C.

(2) Probe Isolation

Often for the sake of simplicity of the experiment, it is not necessary to purify the labeled probe. At some point, the purified probe will improve the electrophoretic results of EMSA. For purification, follow the steps below:

a. For 100 μl of the labeled probe, add 25 μl 5 M ammonium acetate and add 200 μl of absolute ethanol to mix

b. Precipitation at -70 ° C to -80 ° C for 1 hour or at -20 ° C overnight

c. Centrifuge at 12 000 g-16 000 g for 30 min at 4 ° C. Be careful to remove the supernatant

d. Centrifuge at 12 000 g-16 000 g for 1 min at 4 ° C. Carefully remove the residual liquid. Micro-dry precipitation, but not too dry

e. Add 100 μl of TE to completely dissolve the precipitate. The labeled probe should better be used immediately, or no longer than 3 days. The labeled probe can be stored at -20 ° C

(3) EMSA glue preparation

1. Prepare the mold for the inverted leather. You can use conventional molds for irrigation gel electrophoresis, or other suitable molds. The best choice can be filling thin plastic mold, in order to facilitate the subsequent follow-up operation. In order to get better results, you can choose to fill a larger EMSA plastic mold.

2. Prepare 20 ml of 4% polyacrylamide gel according to the following recipe (note that the different proportions of Acr / Bis have little effect on the results).

a. TBE buffer (10X): 1 ml

16.2 ml of distilled water

b. 39: 1 acrylamide / bisacrylamide (40%, w / v): 2 ml

c. 80% glycerol: 625 μl

d. 10% ammonium persulfate: 150 μl

e. TEMED: 10 μl

3. Add the individual solutions in the order above, mix them before adding TEMED, mix immediately after adding TEMED, and immediately add to the molds to avoid bubbles and add combs.

(4) EMSA

1. Set the EMSA binding reaction as follows:

Negative control reaction:

a. Nuclease-Free Water: 7 μl

b. EMSA / Gel-Shift binding buffer (5X): 2 μl

c. nucleoprotein or purified transcription factor: 0 μl

d. labeled probe: 1 μl

e. Total volume: 10 μl

Sample reaction:

a. Nuclease-Free Water: 5 μl

b. EMSA / Gel-Shift binding buffer (5X): 2 μl

c. nucleoprotein or purified transcription factor: 2 μl

d. labeled probe: 1 μl

e. Total volume: 10 μl

Probe cold competition reaction:

a. Nuclease-Free Water: 4 μl

b. EMSA / Gel-Shift binding buffer (5X): 2 μl

c. nucleoprotein or purified transcription factor: 2 μl

d. Unlabeled probe: 1 μl

e. labeled probe: 1 μl

f. Total volume: 10 μl

Mutation Probe cold competition reaction:

a. Nuclease-Free Water: 4 μl

b. EMSA / Gel-Shift binding buffer (5X): 2 μl

c. nucleoprotein or purified transcription factor: 2 μl

d. Unlabeled mutant probe: 1 μl

e. labeled probe: 1 μl

f. Volume: 10 μl

Super-shift reaction:

a. Nuclease-Free Water: 4 μl

b. EMSA / Gel-Shift binding buffer (5X): 2 μl

c. nucleoprotein or purified transcription factor: 2 μl

d. target protein specific antibody: 1 μl

e. labeled probe: 1 μl

f. Total volume: 10 μl

2. Add the reagents in the order described above, mix well before adding the labeled probe, and place at room temperature (20–25 ° C) for 10 minutes to eliminate the non-specific binding of the probe and protein, or let the cold probe give priority to. Then add the labeled probe, mix and place at room temperature (20–25 ° C) for 20 minutes.

3. Add 1 μl of EMSA / Gel-Shift loading buffer (colorless, 10X) and mix immediately. Note: Sometimes bromophenol blue will affect the combination of protein and DNA, it is recommended to try to use colorless EMSA / Gel-Shift loading buffer. If you can not use the colorless loading buffer while loading, you can add a very small amount of blue loading buffer to the colorless sample buffer to observe the blue color.

(5) Electrophoretic analysis

1. Use 0.5XTBE as the electrophoretic solution. Pre-electrophoresis for 10 minutes at 10 V / cm. If there is spare on the hole in the pre-electrophoresis, you can add a small amount of diluted 1X EMSA loading buffer (blue) to see if the voltage is normal.

2. Add a sample that mixes the loading buffer into the sample inlet. Add 10 μl of the diluted 1X EMSA / Gel-Shift loading buffer (blue) to the extra sample wells for observation of electrophoresis.

3. According to 10 V / cm voltage electrophoresis. Make sure that the temperature of the gel does not exceed 30°C. If the temperature is increased, the voltage should be reduced as appropriate. Electrophoresis to the blue dye in EMSA / Gel-Shift loading buffer Bromophenol Blue to the lower edge of glue to stop electrophoresis

4. Cut a thick filter paper similar to or slightly larger than the EMSA glue. Carefully remove the folder with EMSA glue; roughly dry the voltage solution on the edge of the glue with absorbent paper or plain grass paper. Carefully open the top of the two sheets (note: usually choose to remove the silanized piece of glass plate), let the filter paper gradually cover the entire EMSA glue from the side of the EMSA glue; gently press the paper and glue. Gently lift the filter paper after the filter paper was slightly soaked (about less than 1 minute). Put the filter paper side down, flat; cover the EMSA glue with a layer of plastic wrap to ensure that there is no bubble between the plastic wrap and glue.

5. Dry EMSA glue on the Drying equipment. And then test with X-ray film, or with other appropriate equipment.

Collected by Creative BioMart.