CN110904069A - PPK2 protein and application thereof in polyacrylamide gel electrophoresis 35kd standard substance - Google Patents
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Abstract
The invention discloses a PPK2 protein and application thereof as a polyacrylamide gel electrophoresis 35kd standard substance, wherein Nde I and Hind III are utilized to insert a ppK2 gene into a prokaryotic expression vector pET30a, a recombinant expression vector is transferred into an Escherichia coli BL21(DE3) strain through a physical method, IPTG is utilized to induce a target protein PPK2 to perform test expression at 37 ℃, 25 ℃ and 16 ℃, and expression products are identified and analyzed through SDS-PAGE electrophoresis and Blotting; finally, the process is carried out in a batch,amplifying culture is carried out by using 3L of expression bacterial liquid, and the expression product is separated and purified by a Ni-IDA affinity chromatographic column, an anion exchange chromatographic column and a gel filtration chromatographic column in sequence. The results show that: the large intestine prokaryotic expression system can be used at 16 ℃ and the final concentration of 0.2 mM.L‑1The IPTG can be stably and efficiently expressed under induction, the PPK2 protein has specific charged property, exists in a monomer form in a solution, has stable property, is not easy to degrade, has long half-life period, and has an application prospect of being used as a polyacrylamide gel electrophoresis 35kd standard product.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to PPK2 protein and application thereof as a polyacrylamide gel electrophoresis 35kd standard substance.
Background
The protein standard substance has the main function of indicating the molecular weight corresponding to a protein band on protein polyacrylamide gel electrophoresis, is a positive control, and is only convincing if the standard substance is accurate. In addition, the protein standard also has the functions of indicating the success of membrane transfer during Western blot or the electrophoresis degree of the protein on gel, and the like, so that the selection of the correct protein standard is one of the necessary conditions for the success of molecular biological experiments.
Polyphosphate kinase 2 (PPK 2) plays an important role in pathogenicity of a plurality of pathogenic bacteria, and the PPK2 gene is not found in the bodies of human and other metazoans, so that PPK2 is a target point for developing novel antibiotics. However, no related research on the PPK2 protein as a standard product of polyacrylamide gel electrophoresis is reported.
Disclosure of Invention
The invention mainly aims to provide a PPK2 protein, wherein the amino acid sequence of the PPK2 protein is shown as SEQ ID NO: 1 is shown.
The invention also provides a coding gene of the PPK2 protein.
Optionally, the nucleotide sequence of the gene encoding the PPK2 protein is as shown in SEQ ID NO: 2, as shown in the figure:
the invention further provides an expression vector or an expression engineering bacterium containing the PPK2 protein coding gene.
Optionally, the PPK2 protein expression vector is obtained by inserting the coding gene of the PPK2 protein between Nde i and Hind iii cleavage sites of a prokaryotic cell expression plasmid.
Optionally, the prokaryotic cell expression plasmid selects pET30a as a parent expression vector.
The invention also provides application of the PPK2 protein in polyacrylamide gel electrophoresis 35kd standard.
The invention also provides a preparation method of the PPK2 protein, which comprises the following steps:
the recombinant expression plasmid PPK2/pET30a was physically transferred into Escherichia coli BL21(DE3) strain using 0.2 mM. L-1The target protein PPK2 in the final concentration IPTG induced 3L expression strain (liquid) is expressed at 16 ℃, and the concentration can reach 12mg/mL after purification by series chromatography-1The purity was 95%.
The invention has the advantages and beneficial effects that:
1. the PPK2 protein and Western blot electrophoresis standard 10-160 kd (160, 120, 70, 50, 40, 35, 20 and 10) are subjected to SDS-PAGE electrophoretic analysis, and the PPK2 protein band is clear and is just positioned at 35kd, and no other band exists on a lane.
2. The PPK2 protein and common SDS-PAGE electrophoresis standard products 14.4-116 kd (14.4, 18.4, 25, 35, 45, 66.4 and 116) are subjected to SDS-PAGE electrophoresis analysis, and the PPK2 protein band is completely stained and clear, and is located in a lane without other bands and at 35 kd.
3. Anion exchange chromatography and gel filtration chromatography analysis show that PPK2 protein has specific charged property, exists in a monomer form in a solution, has stable property, is not easy to degrade and has long half-life. This is a significant advantage over some commercially available single band protein standards, some of which degrade during use, resulting in no standard band on the gel after electrophoresis is complete.
4. The concentration of 12mg/mL can be purified by using 3L of the expression bacterial liquid as described above-1PPK2 protein with the purity of 95 percent,therefore, the efficiency of expressing the PPK2 protein by adopting an escherichia coli prokaryotic expression system is high, and the method is suitable for preparing the PPK2 protein standard substance by utilizing the system.
Drawings
FIG. 1: map of PPK2/pET30a recombinant expression vector.
FIG. 2: and (3) carrying out whole-plasmid enzyme digestion identification on the PPK2/pET30a recombinant expression vector. C: control (recombinant expression plasmid); PD: nde I and Hind III enzyme digestion of the whole plasmid; m: a DNA marker.
FIG. 3: the expression of the PPK2 protein test was analyzed by SDS-PAGE. M: protein molecular mass standard; c: control (no IPTG addition); t1: inducing for 16h at 16 ℃; t2: inducing for 16h at 25 ℃; t3: induction was carried out at 37 ℃ for 16 h.
FIG. 4: western blot identifies the expression of PPK2 protein at 16 ℃. WM: molecular mass standard of Westernblot protein; t1: induction was carried out at 16 ℃ for 16 h.
FIG. 5: the result was purified by passing PPK2 through Ni-IDA affinity column. M: protein molecular mass standard; s: centrifuging the whole bacteria to obtain supernatant; ie: discharging liquid after the supernatant is incubated with Ni-IDA; ce: washing the effluent liquid; a1: 50mM/L imidazole elution fraction; a2: 100mM/L imidazole eluate fraction; A3-A9: 300mM/L imidazole eluate fraction; nm: PPK2 on Ni column media after elution.
FIG. 6: and (3) purifying the PPK2 protein by anion exchange chromatography.
FIG. 7: SDS-PAGE analysis of anion exchange chromatography purified PPK2 protein. Q21-Q24: a protein collection tube; m: and (3) protein molecular mass standard.
FIG. 8: and (3) purifying the PPK2 protein by gel chromatography.
FIG. 9: SDS-PAGE analysis of gel chromatographically purified PPK2 protein. D18-D23: 18-23 protein collection tubes; m: and (3) protein molecular mass standard.
For a person skilled in the art, other relevant figures can be obtained from the above figures without inventive effort.
Detailed Description
In order to make the technical solution of the present invention better understood, the technical solution of the present invention is further described below with reference to specific examples.
Target genes and plasmids: plasmid pET30a was used as an expression vector (kanamycin resistance, the N-terminus of PPK2 protein only contained 6 XHis tag, which was not excised during purification), and the gene of PPK2 mesh was synthesized by Beijing Okko Spngsheng Biotech Co., Ltd.
The main reagents are as follows: restriction endonucleases (Nde i and Hind iii), SDS-PAGE Marker and DNA Marker were purchased from Thermo corporation, usa; western blot Marker was purchased from Desita Biotechnology (Nanjing) Ltd; the DNA recovery kit and the plasmid small-scale extraction kit are purchased from Tiangen Biochemical technology (Beijing) Co., Ltd; the Bradford protein concentration assay kit was purchased from Biyuntian Biotechnology (Shanghai) Co., Ltd; AKTA protein purifiers, Ni-IDA affinity columns, anion exchange columns HitrapQ HP, and gel filtration chromatography columns Superdex200 Increate 10/300GL were purchased from GE, USA.
Example 1: bioinformatics prediction
After obtaining the amino acid sequence of the PPK2 protein from NCBI database (WP _015420983.1), the PSIPRED website (http:// web. expasy. org/protparam) was used to predict the secondary structure of the PPK2 protein; the basic physicochemical properties of PPK2 were predicted using the ExPASY website (http:// bio if.cs.ucl. ac.uk/psipred /).
Example 2: whole plasmid digestion and sequencing
Utilizing restriction endonucleases Nde I and Hind III to carry out enzyme digestion on a recombinant expression plasmid PPK2/pET30a synthesized by Beijing Odoku ding Biotech Co., Ltd to carry out full-plasmid enzyme digestion identification, wherein a 4216bp strip and 1 1964bp strip are required in the result of a DNA agarose gel electrophoresis experiment; meanwhile, the PPK2/pET30a recombinant expression plasmid is handed to Jinweizhi biotechnology limited to complete the sequencing work of the target gene PPK 2. Finally, the accuracy of the recombinant expression vector was confirmed.
Example 3: PPK2 protein test expression and identification
The recombinant expression plasmid PPK2/pET30a, which was constructed correctly as described above, was transformed into competent cells of Escherichia coli BL21(DE3), and then spread evenly onto LB plates (containing 50. mu.g/ml kanamycin), followed by being placed upside down in an incubator at 37 ℃ overnight. The single clone was selected from the transformed plate, inoculated into 5ml of LB medium (containing 50. mu.g/ml kanamycin), cultured at 37 ℃ in a shaker at 220r/min, and when the culture was carried out to an OD600 value of 0.5 to 0.8, IPTG was added to the test tube culture solution to a final concentration of 0.2mM/L, followed by induction of expression at 16 ℃ and 25 ℃ and 37 ℃ respectively. Centrifuging the induced culture solution at 12000rpm for 5min, removing supernatant, adding 50mM/L Tris-HCl (pH8.5) buffer solution to resuspend the precipitate, adding SDS-PAGE loading buffer solution, heating the sample at 100 deg.C for 10min, and centrifuging to obtain supernatant for SDS-PAGE electrophoresis. Based on the electrophoresis result, the expression condition of the PPK2 protein is further analyzed and identified by Western blot experiment.
Example 4: amplification culture of PPK2 protein
And determining the temperature of the PPK2 protein which is optimally induced and expressed according to the test expression result to be 16 ℃, and carrying out amplification culture by adopting 3L cultured expression bacterial liquid. When the OD600 value of the bacterial liquid is 0.8, the temperature of the bacterial liquid is reduced from 37 ℃ to 16 ℃, IPTG is added to enable the final concentration to be 0.2mM/L, PPK2 is induced at 16 ℃ to express for 16-18 h, and then the bacteria are collected.
Example 5: Ni-IDA affinity chromatography purification
The whole bacteria are ultrasonically cracked by 50mM/L Tris-HCl (pH8.5), 300mM/L NaCl, 20mM/L imidazole containing 1% Triton X-100 and 1mM/L PMSF, and meanwhile, the Ni-IDA affinity chromatography column is balanced by 50mM/L Tris-HCl (pH8.5), 300mM/L NaCl and 20mM/LImidazole buffer solution, then the target protein PPK2 is eluted by imidazole buffer solution with different concentrations, and each eluted component is collected for SDS-PAGE analysis and detection.
Example 6: anion exchange chromatography and gel filtration chromatography
After the PPK2 protein eluate was concentrated using an ultrafiltration tube, it was subsequently purified using an AKTA protein purifier. First, separation and purification were carried out using an anion exchange column (HitrapQ HP), and the collected proteins were subjected to SDS-PAGE electrophoretic analysis based on the position of the peak of the objective protein PPK 2. And concentrating the collected liquid with higher protein purity to 500 mu l, further purifying by using a gel filtration chromatographic column (Superdex200), and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis detection on the collected protein eluate according to the peak position in a gel chromatogram. Mixing and concentrating the PPK2 protein eluent with higher concentration and purity, subpackaging, measuring the concentration by using a Bradford protein concentration measuring kit, and storing in a refrigerator at-80 ℃.
Results
1. PPK2 protein bioinformatics prediction
The basic physicochemical properties of the proteobacteria PPK2 protein (shown in Table 1) are analyzed and β by using website ExPASY (http:// web. ExPASy. org/protparam) statistics, according to the isoelectric point (pI) of the PPK2 in the Table 1 being 5.69, the target protein PPK2 can be separated and purified by an anion exchange chromatographic column by using 50mM/L Tris-HCl buffer solution with pH of 8.5 in the subsequent separation and purification process.
TABLE 1PPK2 basic physicochemical Properties
A PSIPRED website (http:// bio if.cs.ucl. ac.uk/PSIPRED /) is used for predicting the secondary structure of the β Proteobacteria PPK2 protein, the PPK2 does not have a transmembrane domain and a signal peptide, and therefore the PPK2 should be an intracellular soluble protein, and α helix and β folded sheets in the PPK2 are distributed uniformly.
2. PPK2/pET30a whole plasmid restriction enzyme identification
The total length of the PPK2/pET30a recombinant expression vector (as shown in FIG. 1) is 6180bp, restriction endonucleases Nde I and Hind III are used for carrying out full-plasmid cutting identification, and through 1% agarose gel electrophoresis analysis, a 4216bp band and a 1964bp band should be arranged on the same lane. As fig. 2 conforms to the expectations described above.
3. PPK2 protein test expression and identification
The test expression produced PPK2 protein with 6 XHis tag at N-terminal, and the molecular weight of the test expression product induced at 16 deg.C, 25 deg.C, 37 deg.C on SDS-PAGE should be about 35kd according to the molecular weight prediction of PPK2 protein in Table 1 above. As shown in FIG. 3, the expected size is consistent, the expression amount and state of PPK2 protein induced to be expressed at 16 ℃ are more ideal, and the selection of the subsequent amplification culture stage is performed at 16 ℃. To further confirm the above experimental results, Western blot analysis was performed on the test expression cells at 16 ℃ and revealed that the exposure position of the antibody against the N-terminal 6 XHis tag of PPK2 protein was also 35kd (see FIG. 4).
4. Separation of PPK2 protein by Ni-IDA affinity chromatography
And (3) carrying out IPTG induction, ultrasonic crushing and centrifugation on the 3L expression bacterial liquid for amplified culture, then passing through a Ni-IDA affinity chromatographic column, and carrying out SDS-PAGE electrophoresis analysis on the condition that PPK2 protein is eluted by imidazole with different concentrations. As shown in figure 5, the PPK2 protein exists in the supernatant of the whole bacteria after bacteria breaking and centrifugation, the protein solubility is good, the PPK2 protein can be fully eluted by 300mM/L imidazole, the 'column sticking phenomenon' is not generated basically, and the protein purity is higher.
5. Purification of PPK2 protein
The PPK2 protein eluted from the Ni-IDA affinity chromatographic column is firstly purified by using a HitrapQ HP anion exchange column, and the anion exchange chromatogram of the PPK2 protein shows a symmetrical single peak (as shown in figure 6), which indicates that the protein has more uniform charging property. The eluate was then extracted 5. mu.l from the tube Q21-Q24 for SDS-PAGE (FIG. 7). The chromatogram of the PPK2 protein on the gel filtration chromatographic column shows a single symmetrical peak (as shown in FIG. 8), and the elution peak appears at 14.24ml, which indicates that the PPK2 protein exists in a monomer form in the solution. And 5 mu.l of eluate from D18-D23 tube was absorbed for SDS-PAGE (as shown in FIG. 9), and PPK2 protein has high purity and no other impurity proteins. After D18-D23 tube eluent is mixed evenly and concentrated to 60 mul, the protein concentration is measured to be 12mg/ml, and the purity is 95%.
The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.
SEQUENCE LISTING
<110> Tianjin City university of occupation
<120> PPK2 protein and application thereof in polyacrylamide gel electrophoresis 35kd standard substance
<130>1
<160>2
<170>PatentIn version 3.5
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<211>301
<212>PRT
<213>Betaproteobacterium
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Met Thr Ser Lys His Lys Glu Met Ala Glu Trp Tyr Gln Arg Ala Gln
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Glu Glu Ile Leu Asp Ser Met Asp Glu Glu Leu Glu Met Glu Leu Asp
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Asp Asp Arg Leu Ser Gln Asp Gly Gly Ser Ser Ser Ile Ile Pro Arg
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Asn Val Tyr Phe Lys Glu Leu Phe Arg Leu Gln Gly Glu Leu Val Lys
50 55 60
Leu Gln Asp Trp Val Val Glu Asn Lys Leu Lys Val Ala Val Leu Phe
65 70 75 80
Glu Gly Arg Asp Ser Ala Gly Lys Gly Gly Ala Ile Lys Arg Ile Thr
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Gln Arg Leu Asn Pro Arg Val Cys Lys Val Val Ala Leu Thr Ala Pro
100 105 110
Ser Glu Arg Glu Lys Thr Gln Trp Tyr Phe Gln Arg Tyr Val Ser Asn
115 120 125
Leu Pro Ala Gly Gly Glu Ile Val Leu Phe Asp Arg Ser Trp Tyr Asn
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Arg Ala Gly Val Glu Lys Val Met Gly Phe Cys Thr Asp Asp Glu Tyr
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Glu Glu Phe Leu Arg Thr Val Pro Glu Phe Glu Arg Met Ile Ile Arg
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Ser Gly Ile Ile Leu Ile Lys Tyr Trp Phe Ser Ile Ser Asp Asp Glu
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Gln Tyr Asn Arg Phe Met Met Arg Ile His Asp Pro Leu Lys Gln Trp
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Lys Leu Ser Pro Met Asp Leu Asp Ala Arg Arg His Trp Glu Ala Tyr
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Thr Lys Ala Lys Glu Thr Met Leu Glu Arg Thr Asn Ile Pro Glu Ala
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Pro Trp Trp Val Val Ala Ala Asn Asp Lys Lys Lys Ala Arg Leu Asn
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Cys Ile Ser His Leu Leu Asp Gln Ile Pro Tyr Lys Glu Ile Asp His
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Pro Glu Ile Thr Leu Pro Ala Arg Val His Asn Pro Asp Tyr Leu Arg
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Gly Pro Val Pro Lys Glu Met Tyr Val Pro Glu Ile Tyr
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atgacatcaa aacacaaaga gatggctgag tggtatcagc gtgcacaaga agaaatcttg 60
gatagcatgg atgaagagct cgaaatggag ctcgatgatg atcgcctgtc gcaggacggt 120
ggcagtagct ctattattcc gcgaaacgtt tactttaagg agttatttcg tcttcaaggt 180
gaattagtaa agctccaaga ttgggttgtt gaaaataaac tcaaggtggc tgtgttgttt 240
gaaggccgcg attctgcggg taagggcgga gctatcaaac gtattaccca gcgactcaat 300
ccccgtgttt gtaaagtagt cgcgctgacc gcacctagcg aacgtgaaaa gacccagtgg 360
tattttcagc gttatgtatc caatttaccc gcaggcggtg aaatcgttct ctttgatcgc 420
agctggtata accgggccgg tgttgagaag gtcatgggat tttgtactga tgatgagtac 480
gaagaatttt tacgcacggt tcccgagttt gagcgcatga tcattcgctc tgggattata 540
ttaattaaat actggttctc aatttcagat gatgagcagt acaaccgctt catgatgcgt 600
atccatgatc ctttgaagca gtggaagtta agcccaatgg atctcgatgc acgtcgtcac 660
tgggaagcct acacaaaggc aaaagagacc atgttagagc gcaccaatat ccctgaggca 720
ccatggtggg ttgttgctgc aaacgataaa aagaaggcgc gcttaaactg catctcccat 780
ctacttgatc agattcctta caaagagatt gatcatccag aaattactct gccagcccgt 840
gtccataatc cagactactt gaggggtccg gttcccaagg aaatgtacgt ccccgagatc 900
tactga 906
Claims (5)
1. A PPK2 protein, characterized in that: the amino acid sequence of the PPK2 protein is shown as SEQ ID NO: 1 is shown.
2. A gene encoding the PPK2 protein according to claim 1, wherein said gene comprises at least one of: the nucleotide sequence of the encoding gene of the PPK2 protein is shown as SEQ ID NO: 2, respectively.
3. An expression vector or an expression engineering bacterium comprising the gene encoding the PPK2 protein according to claim 2.
4. A method of producing the PPK2 protein of claim 1, wherein the PPK2 protein comprises:
the recombinant expression plasmid PPK2/pET30a was physically transferred into Escherichia coli BL21(DE3) strain using 0.2 mM. L-1The final concentration of IPTG induces the expression of the target protein PPK2 at 16 ℃, and the concentration reaches 12mg/mL after the purification by series chromatography-1The purity was 95%.
5. Use of the PPK2 protein of claim 1 as a polyacrylamide gel electrophoresis 35kd standard.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115010789A (en) * | 2022-06-20 | 2022-09-06 | 湖州申科生物技术有限公司 | Fluorescent polypeptide for quality control of electric focusing in protein dimensional electrophoresis and preparation method thereof |
| CN115216457A (en) * | 2021-04-15 | 2022-10-21 | 华东理工大学 | Extremophilic polyphosphate kinase and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105368838A (en) * | 2015-12-21 | 2016-03-02 | 山东建筑大学 | Microlunatus phosphovorus engineering strain capable of efficiently accumulating polyphosphate and application thereof |
| WO2018203482A1 (en) * | 2017-05-01 | 2018-11-08 | 株式会社カネカ | Production method for substance using atp |
| CN110132680A (en) * | 2019-06-05 | 2019-08-16 | 山东大学 | A kind of acidic polysaccharose electrophoresis standard items and its preparation method and application |
-
2019
- 2019-12-04 CN CN201911225829.0A patent/CN110904069A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105368838A (en) * | 2015-12-21 | 2016-03-02 | 山东建筑大学 | Microlunatus phosphovorus engineering strain capable of efficiently accumulating polyphosphate and application thereof |
| WO2018203482A1 (en) * | 2017-05-01 | 2018-11-08 | 株式会社カネカ | Production method for substance using atp |
| CN110132680A (en) * | 2019-06-05 | 2019-08-16 | 山东大学 | A kind of acidic polysaccharose electrophoresis standard items and its preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| HAO,Z等: "Beta proteobacterium CB chromosome, complete genome, Accession ID: CP004348", 《GENBANK数据库》 * |
| 黄金玲等: "多聚磷酸相关蛋白结构及生物学功能", 《中国生物化学与分子生物学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115216457A (en) * | 2021-04-15 | 2022-10-21 | 华东理工大学 | Extremophilic polyphosphate kinase and application thereof |
| CN115010789A (en) * | 2022-06-20 | 2022-09-06 | 湖州申科生物技术有限公司 | Fluorescent polypeptide for quality control of electric focusing in protein dimensional electrophoresis and preparation method thereof |
| CN115010789B (en) * | 2022-06-20 | 2023-10-20 | 湖州申科生物技术股份有限公司 | Fluorescent polypeptide for medium electrofocusing quality control in protein dielectrophoresis and preparation method thereof |
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