Experimental method principle

Gel migration or electrophoretic mobility shift experiment (EMSA-electrophoretic mobility shift assay) is a technique to study the interaction between DNA binding proteins and their related DNA binding sequences, which can be used for qualitative and quantitative analysis. This technique was originally used to study DNA binding proteins, and has been used to study the interaction of RNA binding proteins with specific RNA sequences.

Usually, the purified protein and crude cell extract are incubated with 32P isotope-labeled DNA or RNA probe, and the complex and unbound probe are separated on non-denatured polypropylene gel electrophoresis. DNA-complexes or RNA-complexes move slower than unbound probes. Isotope-labeled probes may be double-stranded or single-stranded depending on the binding protein studied. When detecting DNA binding proteins such as transcriptional regulators, purified proteins, partially purified proteins, or nuclear cell extracts can be used. When detecting RNA-binding proteins, depending on the position of the RNA-binding protein of interest, purified or partially purified proteins can be used, as can nuclear or cytoplasmic cell extracts. In competition experiments, DNA or RNA fragments and oligonucleotide fragments (specific) containing protein binding sequences and other non-related fragments (non-specific) are used to determine the specificity of DNA or RNA binding proteins. In the presence of competitive specific and non-specific fragments, specific binding is determined according to the characteristics and strength of the complex.

Experimental Materials

[γ-32P] ATPT4 polynucleotide kinase Nuclease-FreeWaterT4 polynucleotide kinase buffer ammonium acetate TE absolute ethanol TBE buffer double distilled water methylene bisacrylamide acrylamide glycerol ammonium persulfate TEMED (tetramethyl (Ethylenediamine) EMSA Gel-Shift binding buffer bromophenol blue

Instruments, consumables

Water bath pot PCR instrument centrifuge electrophoresis instrument electrophoresis tank

[γ-32P] ATPT4 polynucleotide kinase Nuclease-FreeWaterT4 polynucleotide kinase buffer ammonium acetate TE absolute ethanol TBE buffer double distilled water methylene bisacrylamide acrylamide glycerol ammonium persulfate TEMED (tetramethyl (Ethylenediamine) EMSA Gel-Shift binding buffer bromophenol blue

Instruments, consumables

Water bath pot PCR instrument centrifuge electrophoresis instrument electrophoresis tank

[γ-32P] ATPT4, polynucleotide kinase, Nuclease-FreeWaterT4, polynucleotide kinase buffer,

Ammonium acetate, TE, absolute ethanol, TBE buffer, redistilled water, methylene bisacrylamide, acrylamide, glycerin,

Ammonium persulfate, TEMED (tetramethylethylenediamine), EMSA Gel-Shift binding buffer, bromophenol blue, water bath, PCR instrument, centrifuge, electrophoresis instrument, electrophoresis tank

Experimental procedure

First, the label of the probe

1. Set the probe labeling reaction system as follows:

(1) Probe to be labeled (1.75 pmol / microliter): 2 microliters.

(2) T4 Polynucleotide Kinase Buffer (10X): 1 microliter.

(3) Nuclease-Free Water: 5 microliters.

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

(5) T4 Polynucleotide Kinase (5-10 u / microliter): 1 microliter.

(6) Total volume 10 μl

(7) Add various reagents in sequence according to the above reaction system. After adding the isotope, mix Vortex, then add T4 Polynucleotide Kinase and mix.

2. Using a water bath or PCR machine, react at 37 ° C for 10 minutes.

3. Add 1 μl of probe labeling stop solution, mix well to stop the probe labeling reaction.

4. Add 89 microliters of TE and mix well. At this time, a small amount of probe can be used to detect the efficiency of the label. The efficiency of labeling is usually more than 30%, that is, more than 30% of the total radioactivity is labeled on the probe. For simplicity of experiment, it is usually unnecessary to measure the labeling efficiency of the probe.

5. The labeled probe is best used immediately, and the longest use time is generally not more than 3 days. The labeled probe can be stored at -20 ° C.

Second, the purification of the probe

Usually, for simplicity of experiment, it is not necessary to purify the labeled probe. In some cases, the purified probe will improve the EMSA electrophoresis results. For purification, you can follow the steps below:

1. For 100 microliters of labeled probe, add 1/4 volume of 25 microliters of 5 M ammonium acetate, then add 2 volumes of 200 microliters of absolute ethanol, and mix.

2. Precipitate at -70 ° C to -80 ° C for 1 hour or overnight at -20 ° C.

3. Centrifuge at 12,000 g-16000 g for 30 minutes at 4 ° C. Carefully remove the supernatant and never touch the sink.

4. Centrifuge at 12,000 g-16000 g for 1 minute at 4 ° C. Carefully suck up residual liquid. Allow the pellet to dry slightly, but not excessively.

5. Add 100 µl of TE to completely dissolve the precipitate. The labeled probe is best used immediately, and the longest use time is generally not more than 3 days. The labeled probe can be stored at -20 ° C.

3. Preparation of EMSA glue

1. Prepare the pouring mold. A conventional mold for pouring protein electrophoresis gel, or other suitable molds may be used. It is best to choose a mold that can be filled with thinner glue to facilitate subsequent operations such as dry glue. For better results, you can choose a mold that can be filled with larger EMSA glue.

2. Prepare 20 ml of 4% polyacrylamide gel according to the following formula (Note: The use of different ratios of Acr / Bis such as 29: 1 has little effect on the results).

(1) TBE buffer (10X): 1 ml.

16.2 ml of redistilled water.

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

(3) 80% glycerin: 625 microliters.

(4) 10% ammonium persulfate: 150 microliters.

(5) TEMED: 10 microliters

3. Add each solution in the above order, mix well before adding TEMED, mix immediately after adding TEMED, and immediately add it to the mold for making rubber. Avoid air bubbles and add combs. If you find that it is very easy to form bubbles, you can silanize a glass plate made of glue.

4. EMSA combination reaction

1. Set the EMSA binding reaction as follows

Negative control reaction:

(1) Nuclease-Free Water: 7 microliters.

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

(3) Nuclear protein or purified transcription factor: 0 μl.

(4) Labeled probe: 1 microliter.

(5) Total volume: 10 microliters.

Sample reaction:

(1) Nuclease-Free Water: 5 microliters.

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

(3) Nuclear protein or purified transcription factor: 2 microliters.

(4) Labeled probe: 1 microliter.

(5) Total volume: 10 microliters.

Probe cold competition reaction:

(1) Nuclease-Free Water: 4 microliters.

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

(3) Nuclear protein or purified transcription factor: 2 microliters.

(4) Unlabeled probe: 1 microliter.

(5) Labeled probe: 1 microliter.

(6) Total volume: 10 microliters.

Cold competition reaction of mutation probe:

(1) Nuclease-Free Water: 4 microliters.

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

(3) Nuclear protein or purified transcription factor: 2 microliters.

(4) Unlabeled mutation probe: 1 microliter.

(5) Labeled probe: 1 microliter.

(6) Volume: 10 microliters

Super-shift response:

(1) Nuclease-Free Water: 4 microliters.

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

(3) Nuclear protein or purified transcription factor: 2 microliters.

(4) Target protein specific antibody: 1 microliter.

(5) Labeled probe: 1 microliter.

(6) Total volume: 10 microliters.

2. Add various reagents in sequence according to the above sequence, mix well before adding the labeled probe, and place at room temperature (20-25 ° C) for 10 minutes to eliminate possible non-specific binding of the probe and protein, or Let the cold probe react first. Then add the labeled probe, mix well, and place at room temperature (20-25 ° C) for 20 minutes.

3. Add 1 μl of EMSA / Gel-Shift loading buffer (colorless, 10X), mix immediately and load immediately. Note: Sometimes bromophenol blue will affect the binding of protein and DNA, it is recommended to use colorless EMSA / Gel-Shift loading buffer as much as possible. If you feel that you can not load the sample with the colorless loading buffer, you can add a very small amount of blue loading buffer into the colorless loading buffer until you can observe the blue color.

5. Electrophoresis analysis

1. Use 0.5XTBE as the electrophoresis solution. Pre-electrophoresis at a voltage of 10V / cm for 10 minutes. If there are spare loading wells during pre-electrophoresis, a small amount of diluted 1X EMSA loading buffer (blue) can be added to observe whether the voltage is running normally.

2. Add the sample mixed with loading buffer to the loading well. Add 10 μl of diluted 1X EMSA / Gel-Shift loading buffer (blue) to one of the extra sample wells to observe the progress of electrophoresis.

3. Electrophoresis at a voltage of 10 V / cm. Make sure that the temperature of the glue does not exceed 30 ° C. If the temperature increases, the voltage needs to be reduced appropriately. Electrophoresis to the blue dye bromophenol blue in the EMSA / Gel-Shift loading buffer to 1/4 of the lower edge of the gel to stop electrophoresis.

4. Cut a piece of thicker filter paper that is similar to or slightly larger than the size of EMSA glue. Carefully remove the rubber sheet with EMSA glue, and use absorbent paper or ordinary straw paper to roughly dry the voltage solution on the edge of the rubber sheet. Carefully open the top of the two rubber plates (Note: usually choose to remove the silanized glass plate first), gradually cover the filter paper from the side of the EMSA glue, and gently press the filter paper and the glue tightly . After the filter paper is slightly soaked by the glue (about less than 1 minute), gently lift the filter paper, then the EMSA glue will be lifted together by the filter paper. Put the filter paper side down and lay it flat. Cover the EMSA glue with a layer of cling film to ensure that there are no air bubbles between the cling film and the glue.

5. Dry the EMSA glue on the dry glue instrument. Then use X-ray film to test, or use other appropriate equipment.

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