How to design EMSA experimental detector?

Gel migration experiment

1) What is gel migration or electrophoretic mobility experiment?

Gel migration or electrophoretic migration experiment (EMSA) 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 now it has been used to study the interaction between RNA binding proteins and specific RNA sequences.

Generally, purified protein, cell crude extract and 32P isotope-labeled DNA or RNA probe are incubated together, and the complex and unbound probe are separated on non-denatured polypropylene gel electrophoresis. DNA complexes or RNA complexes move more slowly than unbound probes. Isotopically labeled probes can be double-stranded or single-stranded depending on the binding protein studied. When detecting DNA binding proteins such as transcription regulatory factors, purified proteins, partially purified proteins or nuclear extracts can be used. When detecting RNA-binding proteins, purified or partially purified proteins or nuclear or cytoplasmic cell extracts can be used according to the location of the target RNA-binding proteins. In competitive experiments, DNA or RNA fragments and oligonucleotide fragments containing protein binding sequences (specificity) and other unrelated fragments (nonspecific) are used to determine the specificity of DNA or RNA binding proteins. In the presence of competitive specific and nonspecific fragments, the specific binding is determined according to the characteristics and strength of the complex.

2) What reagents are needed for such an experiment?

The binding proteins needed for gel migration experiments can be derived from purified or partially purified proteins or crude nuclear and cytoplasmic extracts. Isotopically labeled DNA or RNA must also be prepared. Generally, DNA nucleotide probes are labeled with g-32P and T4 polynucleotide kinase, and isotopically labeled RNA is synthesized in vitro with phage RNA polymerase and isotopically labeled nucleotides. Promega's riboprobe? /sup system (a, b) (catalog numbers P 1420, P 1430, P 1440, P 1450, P 1460) can be used to synthesize isotopically labeled RNA in vitro, and After the binding protein interacts with the isotope-labeled probe, the complex is separated on the non-denatured polypropylene gel electrophoresis, and then the gel is dried and autoradiographed, or the gel is used together with the phosphorescent image? /sup analysis.

3) What reagents are provided by the gel migration experiment system?

Promega company provides a gel migration experimental system for detecting DNA binding proteins, which can be used as a positive control for such experiments. The system includes target oligonucleotide, control DNA binding protein, binding buffer and reagents needed for oligonucleotide probe end labeling. The core system (catalogue number E3050) includes the Escherichia coli extract (AP2 extract) containing recombinant AP2 protein and the homologous oligonucleotide of AP2. AP2 extract is extracted from Escherichia coli expressing AP2 protein. In addition, the core system also contains SP 1 homologous oligonucleotide, gel migration binding buffer (5'), and HeLa nuclear extract which can be used in 20 controlled experiments. The whole system (catalog number E3300) contains five other double-stranded oligonucleotides, which are homologous sequences of the binding sites of AP 1, OCT 1, CREB, NF-kB and TFIID. These oligonucleotides can be used as specific probes after terminal labeling or as non-specific probes in competitive experiments. For more information, please refer to the gel migration experiment technical manual TB 1 10.

4) What factors need to be optimized for the success of gel migration experiment?

The gel migration experiment is simple and fast in theory, but in order to successfully carry out the gel migration experiment, some parameters need to be optimized, which are mainly influenced by the source of binding protein and the characteristics of probe binding site. The following factors need to be optimized: the preparation of extraction solution (nuclease and phosphatase pollution will degrade the probe), the concentration of binding protein, probe concentration, nonspecific probe concentration, the formula and pH of buffer, the characteristics and electrophoresis conditions of polypropylene gel electrophoresis, incubation time and temperature, carrier protein, whether there are auxiliary factors (such as metal ions such as zinc and cadmium, or hormones). In short, the total reaction volume should be the smallest (20ul). In order to meet the general requirements, the binding buffer contains 4% glycerol, 1mmgcl2, 0.5mm EDTA, 0.5mm DTT, 50mm NaCl,10mtris-HCl (pH 7.5) and 0.05mg/ml poly (di: DC)? DI:dC, or10mhepes (pH 7.9), 50mmkcl, 1mm DTT, 1mm EDTA, 10% glycerol, 0.05mg/ml poly (DI:dC)? DI:dC can be used as the beginning of optimization experiment. For more information, please refer to the gel migration experiment technical manual TB 1 10.

5) What homologous polynucleotide primers are provided, and what are the sequence sources of these primers?

There are many kinds of dsDNA probes, including homologous binding sites of various transcription regulatory factors. The following table lists the available probes and the sources of these primer sequences.

Transcriptional regulator probe

Source of probe name directory number sequence (winding) sequence

Sp 1e323 1, e32325 ′-attcga tcgggggggcg-3 ′ SV40 promoter (1)

AP1E 3201E 32025 ′-CGCTTGATATGCGGAA-3 ′ Collagenase Gene TRE(2)

Ap2e3211e32125'-gatcgaact GAC CGCC CCGGT-3' human metallothionein II a(3)

Gene NF-KB E329 1, E3292, 5'-AGT TGA GGG GAC TTT CCC GGC-3' mouse Igk light chain gene (4)

Oct1e 3241e 32425 ′-tgtcga ATG CAA ATC act aga a-3 ′ ig heavy chain gene (5)

CREB E 3281E 32825 ′-agagat TGC CTG ACG TCA GAC AGC Tag-3 ′ Rat Growth Hormone Inhibitory Gene (6)

Tfiid e 3221e 3222 5'-GCA gag cat ATA aggtga ggtaga-3' beta-1globulin promoter.

Only the upper chain sequence is listed, the probe is double-stranded, and the lower chain sequence is paired with the upper chain sequence. The sequences shown in bold are derived from specific gene sequences, and the sequences bound by transcription regulatory factors are shown by the following lines. Generally speaking, the surrounding nucleotides are arbitrary.

What important factors should be considered in the selection of DNA probes?

The length of the target DNA should be less than 300bp, so as to facilitate the electrophoretic separation of unbound probes and protein DNA complexes. Double-stranded synthetic oligonucleotides and restriction enzyme fragments can be used as probes in gel migration experiments. If the target protein has been identified, a short oligonucleotide fragment (about 25bp) is used so that the binding sites can be distinguished from those of other factors. Long restriction fragments can be used to locate the protein binding site in the putative promoter/enhancer region. Subsequently, DNaseI blot can be used to analyze the specific region bound by protein at the level of DNA sequence.

7) What complexes can be formed with the following transcription regulatory factors and HeLa nuclear extracts: ap 1, AP2, CREB, nfkb, oct 1, sp 1, tfiid, tfiib.

When HeLa nuclear extract is used as the source of binding protein, every 1 transcription regulatory factor binds to its related DNA homologous sequence to form a characteristic binding form. Each transcription regulatory factor is described below, including the recognized homologous sequence, the size of the factor, the specific binding conditions and the number of complexes that can be formed with HeLa nuclear extract.

1.ap 1: ap 1 (activator protein 1) is a transcription regulator, and its binding homologous sequence is 5'-TGA gtca-3'. When the binding site of AP 1 exists in the promoter region of genes, these genes can be induced, for example, protein kinase C (2 2,7) can be induced by phorbol ester. In cells, AP 1 forms homodimer of c-Jun or Jun-related protein, or heterodimer of C-Jun or Jun-related protein and c-Fos or Fos-related antigen (Fras). Fos protein itself can not form homodimer and can not bind to AP 1 binding site alone. C-Jun protein is a 40kDa monomer protein, which forms homodimer through leucine zipper. In HeLa cells, the main form of AP 1 is the homodimer of c-Jun protein. In the gel migration experiment, a specific complex was formed. When determining the activity of AP 1 as a gel migration experiment, in addition to the basic solution components, 0.0 1mg/ml poly (dI:dC)? Di: DC), add 100 ug BSA and 5 mm DTT to the binding buffer. If purified protein is used, 1-2ug protein is used to detect the migration complex.

2.AP2: AP2 is a transcription regulator, which can be used as TPA- and cAMP- inducing factors (10) respectively. It is a 52 kDa protein, and the recognized homologous sequence is 5'-CCCGGC-3' or 5'-GCCCNNGGC-3'(3). This factor is particularly sensitive to retinoic acid and may play an important role in morphogenesis. HeLa nuclear extract and AP2 homologous DNA probe form a specific complex. When the purified protein is used in gel migration experiments, 20-50ng of protein is used.

3.CREB: CREB is a 37 kDa transcription regulator, which recognizes the homologous sequence of 5 ′-T (g/t) ACGTCA-3 ′ DNA (6, 1 1) responding to cAMP. Contains leucine zipper structure to form homodimer, and the related basic domain is homologous to c-JunDNA binding domain. When HeLa nuclear extract is used, it can form a complex with CREB homologous sequence.

4.NF-KB: NF-KB was originally identified as binding to the enhancer of immunoglobulin K light chain in B cells. However, it was later found in the cytoplasm of non-B cells that a complex of NF-kB and IkB was formed. NF-kB originally isolated from DNA binding protein complex is a heterodimer consisting of p65(RelA) and p50. Other isolated monomers include p49 (also called p52), p75 (c-rel) and p68 (relb). Monomers p65, p68 and p75 play a trans-activation role. P50 and p49(p52) monomers have DNA binding activity, but only a small amount of trans activation. It is reported that p49 and NF-kB monomer p65 form a heteroduplex with transcription activity, which is similar to p50/ p65 heteroduplex. P49/ p65 and p50/ p65 heterodimers are regulated in cytoplasm by an inhibitor called IkBa/MAD-3. The combination of IkB and p65 monomer prevents the localization in the nucleus and the combination of DNA. In vitro, high concentration of p65 can form homodimer, which can weakly bind to DNA. Poly (dI:dC) can inhibit this reaction (14). P49 and p50 can also form homodimer, but the concentration in cells is very low. Generally, in the gel migration experiment of NF-kB, there are 0.28 pmol of NF-kB9 oligonucleotide (pH 7.9), 50 mm MKCl, 0.2 mm EDTA, 2.5 mm DTT, 65,438+00% glycerol and 0.05% NP- in a 20ul reaction volume. When using purified protein, 250-300ng is enough to form gel migration complex, and 10ug of HeLa core extract is needed. The gel migration complex was incubated at room temperature for 30 minutes. The gel migration complex was separated by 50mMTris(pH8.3) and 38mM glycine in 7% polyacrylamide gel electrophoresis. The sample solution containing coomassie blue and xylene blue pigment can only be added to the negative control reaction, because these two pigments will aggravate the dissociation of NF-kB complex. When HeLa nuclear extract is used as a binding protein source, two sequence-specific gel migration complexes can be formed, namely p50/p50 homodimer and p50/ p65 heterodimer. Four sequence-specific gel migration complexes (p49/P49, p50/P50, P50/p65, p49/ p65) can be detected in cells expressing P49, P50 and P65. If there is a high concentration of P65, a small amount of p65/p65 can be detected. The following reagents can enhance the binding of NF-kB in vitro: mM GTP, ATP, spermine, spermidine, barium or calcium ion, ng Co+3(NH3)6( 12).

5.OCT 1: OCT 1 is a member of OCT transcription regulator family, which obviously exists widely in mammalian cells (5). POU domain includes POU box and homologous domain. When HeLa core extract is used, the sequence homologous gel migration complex formed with OCT 1 homologous probe can be detected.

6.Sp 1: Sp 1 is an O- glycosylation transcription regulator, which recognizes the homologous sequence 5 ′-ggggggggg c-3 ′ (1) with a length of1. The core recognition sequence is 5'-GGGGGGGGGGG-3'. Sequences similar to the core sequence usually exist in promoters. An example is the early promoter of SV40, which is the first promoter that can bind to SP 1. According to different glycosylation, its molecular weight is 95- 105kDa, and three zinc fingerprints of DNA binding domain determine the specificity of the sequence. HeLa nuclear extract and SP 1 homologous probe form a specific gel migration complex.

7.tfiid/TFIIB: tfiid and TFIIB are basic transcription regulators, which participate in the basic transcription of RNA polymerase II promoter (16). TFIID forms specific DNA binding with TATA region of eukaryotic promoter. TFIID is composed of several proteins, among which TATA domain binding protein (TBP) is involved in the binding of TATA sequence. Other protein components of TFIID are called TBP correlation factors (TAFs). A weak gel migration band can be obtained by using TFID probe oligonucleotide and HeLa nuclear extract, but it is difficult to identify it as TFID sequence-specific gel migration complex. The gel migration experiment of purified recombinant TBP is difficult, partly because TBP has a strong positive charge, which makes it difficult for TBP/DNA complex to enter the gel. The purified TBP can't combine with DNA( 17) after forming dimer. Therefore, the formed dimer can participate in DNA binding. TFIIB does not bind to DNA alone, but after binding to TFIID, it enhances the binding to DNA.

After TFIIB binds to the pre-start complex, RNA polymerase II and TFIIF bind to the transcription initiation region. Therefore, TFIIB plays an important role in the formation of pre-startup complex. When the purified TFIID is used in gel migration experiments, it is unnecessary to add poly (dI-dC) to the binding reaction. The binding buffer contains 10% glycerol, 20 mm tris (pH 8.0), 10 mm MgCl2, 2 mm DTT and 89 mm KCl. Poly (dG:dC) can be added to the gel binding experiment of TFIID. DG:dC. The complex was separated by non-denaturing polyacrylamide gel electrophoresis. The gel composition was 0.5'TBE, 6% polyacrylamide gel (19: 1 acrylamide: dichotomy), 4 mmg cl 2, 0.02%NP-40, and the electrophoresis buffer composition was 0.5'. When studying the complex containing TFIID and TFIIB, MgCl2 should be removed from binding buffer, gel and electrophoresis buffer. These are general requirements. When using different cell extracts, TFIID and TFIIB to form a complex for gel migration experiment, many factors should be optimized to achieve ideal conditions.

8) How many protein or extracts and labeled DNA probes are used in the gel migration experiment?

For each specific binding protein and probe, it is necessary to optimize the purified protein, partially purified protein and crude nuclear extract used. Generally, the dosage of purified protein is between 20 and 20-2000ng, and the equimolar ratio of protein to DNA can be adjusted to 5 times that of DNA. For the crude nuclear extract, 1-20ug protein is needed to form a specific complex. The amount of probe added in the reaction is 50,000-200,000 CPM 32 P labeled probe (high specific activity), and the reaction volume is 65,438+0-5 UL. This is equivalent to 10-50 mmol of DNA probe. The probe should be stored at -20℃ to prevent degradation, and must be used within 1-2 weeks after synthesis or labeling. Both probes and binding proteins should avoid repeated freezing and thawing.

9) Can protein be prepared by in vitro translation?

Promega did not carry out such tests on all transcription regulatory factors. Usually, wheat germ extract is used for in vitro translation of mammalian transcription factors or DNA binding proteins, and rabbit reticulocyte lysate may contain endogenous mammalian transcription factors or DNA binding proteins. But what about TNT /sup & gt； The transcription regulator AP 1(c-Jun) was translated with rabbit reticulocyte lysis system (A, B, C, D) and tRNA labeled with biotin beyond TM (catalog number E320 1) to prepare AP 1 homologous oligonucleotide (catalog number E320 1). TNT？ /sup & gt； T7-coupled wheat germ extracts (b, c, d, e) translated c-Rel in vitro. This protein-specific probe for migrating immunoglobulin K light chain enhancer. Adding MAD-3 (a member of IKB family) translated into c-Rel binding reaction in vitro can interfere with its interaction.

10) poly (dI:dC)? DI:dC), what are the functions of non-specific competitive DNA and specific competitive DNA?

It consists of inosine and cytosine. Because of its special structure, it can inhibit the nonspecific binding of protein with labeled probes and avoid false complexes. Adding poly (dI:dC) to the gel migration reaction? DI:dC) can inhibit the binding of other DNA binding proteins in crude nuclear extract, such as the nonspecific binding of transcription regulatory factors. When the purified protein is used in gel migration reaction, it is unnecessary to add poly (dI:dC)? DI:dC), if added, the final concentration used in ordinary reaction will not exceed 50- 100ng. For nuclear extract, use 1 ug poly (dI:dC) for every 2-3ug of nuclear extract? DI:dC). In order to determine the specificity of the formed complex, the competitive experiment of binding reaction was carried out with or without increasing non-radioactive specific competitive DNA or non-specific competitive DNA. Generally speaking, non-radioactive specific DNA is unlabeled DNA probe, non-specific competitive DNA, which has the same length composition as DNA probe, but different sequence. The complex that can compete with non-radioactive specific DNA but not with non-specific competitive DNA indicates the specific binding of target protein and isotope labeled probe. Nonspecific binding can be competed by specific DNA and nonspecific competitive DNA. Poly (dI:dC) in binding solution? DI:dC) needs to be optimized, but 0.05mg/ml is generally used. The dose of non-radioactive (specific or non-specific) competitive DNA also needs to be optimized or titrated, but the competitive DNA is usually 10- 1000 times (w/w) of the isotope labeled probe. Other types of competitive DNA, such as calf thymus DNA, cannot be used for gel migration reaction, and they will carry the binding site of the target protein.

1 1) What gel conditions are used to separate protein/probe complexes from free probes?

Binding protein or crude core extract is combined with target probe, and protein/probe complex and free probe can be separated by denaturing polyacrylamide gel electrophoresis. The concentration of polyacrylamide is generally 6% (30: 1 acrylamide: binary), and high concentration or low concentration can be used under certain conditions. PH, the concentration of polyacrylamide and the ratio of acrylamide to diblock acrylamide will affect the migration of the complex in the gel. The voltage of10-15v is used for most protein, and the voltage of short-time high voltage (30-35v) is used for rapidly dissociated protein. TBE and TAE used in electrophoresis must be newly prepared without precipitation. The low ionic strength and "box effect" of acrylamide matrix contribute to the stability of the complex. TGE buffer (12.5mMTris, pH8.3, 95mM glycine, 0.5mMEDTA) can also be used for unstable protein /DNA complexes. Binding and electrophoresis experiments can be carried out at 40℃ to prevent the dissociation of unstable complexes and probes. Pigments in the sample solution will lead to the dissociation of unstable complexes, so the sample solution without Coomassie Blue and Xylene Blue should be used. When the band pattern is not tight and tailing occurs, it shows that the compound is dissociated. The gel must be completely polymerized to avoid streaking. If the complex does not enter the gel, it indicates that protein or probe is used excessively, or the salt concentration is too high for the reaction. There are no free probes or complexes in the test strip containing the extract, but there are probes in the test strip containing only probes, which indicates that the extract is contaminated by nucleic acid or phosphatase, so corresponding inhibitors should be added in the extract and binding reaction. At present, protein/probe complexes can be separated by high-strength agarose gel (metaphor? /sup/ agarose)

12) How to determine the existence of a protein in a specific compound?

Partially purified protein or crude nuclear extract and specific probe can form one or several specific protein complexes. The existence of multiple complexes indicates protein degradation, and protease inhibitors should be added in the solution of extract preparation and in the binding reaction. It may be difficult to determine the characteristics of protein in the complex, but there are some methods to study it. If there is an antibody against the target protein, a supermigration experiment can be carried out, and the antibody binds to the protein in the protein/probe complex to delay the migration of the complex and form supermigration. Incremental antibody was added in the binding reaction. The antibody may be added to the protein after reacting with the probe, or the extract may be combined with the antibody before adding the probe. According to the specific antigenic determinant of antibody, the former is beneficial to the formation of supermigration complex, while the latter prevents the formation of complex, resulting in the decrease of the strength of original complex. In most experiments, the molar ratio of antibody to protein is 1: 1, and then the amount of antibody is increased. When purified protein is present, they can be compared with the experimental band migration complexes. In addition to supermigration experiments, the characteristics of protein in the complex can also be analyzed by ultraviolet cross-linking and label transfer. After the uniformly labeled probe was incubated with the nuclear extract, the complex was crosslinked by ultraviolet irradiation, and then the unprotected probe was degraded by DNase. A uniformly labeled probe is needed, because deoxyribonuclease will remove the label from the end-labeled probe. Protein crosslinked by several protected nucleotides was separated by denaturing polyacrylamide gel electrophoresis, dried and autoradiographed. The molecular weight of the binding protein can be compared with the standard molecular reference. Protein blot analysis can also be performed on complexes with antibodies to target proteins (20). If protein binds to a specific sequence of a DNA probe, it can be characterized by a competitive oligonucleotide containing a conserved binding sequence and a mutant. Site-directed mutation can also be used to change the binding sites of conserved sequences to study the formation of complexes.

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