Enzymatic Synthesis of Chimeric DNA Oligonucleotides by in Vitro Transcription with dTTP, dCTP, dATP, and a pair of’-Fluoro Modified dGTP
Environment friendly methods to supply single-stranded DNA are of nice curiosity for various purposes in molecular biology and nanotechnology. Within the current examine, we chosen T7 RNA polymerase mutants with lowered substrate specificity to make use of an in vitro transcription response for the synthesis of chimeric DNA oligonucleotides, both individually or in swimming pools. We carried out in vitro evolution based mostly on fluorescence-activated droplet sorting and recognized mutations V783M, V783L, V689Q, and G555L as novel variants resulting in relaxed substrate discrimination.
Transcribed chimeric oligonucleotides have been examined in PCR, and the standard of amplification merchandise in addition to constancy of oligonucleotide synthesis have been assessed by NGS. We concluded that enzymatically produced chimeric DNA transcripts comprise considerably fewer deletions and insertions in comparison with chemically synthesized counterparts and may efficiently function PCR primers, making the advanced enzymes superior for easy and low cost one-pot synthesis of a number of chimeric DNA oligonucleotides in parallel utilizing a plethora of premixed templates.
1,3-Diketone-Modified Nucleotides and DNA for Cross-Linking with Arginine-Containing Peptides and Proteins
Linear or branched 1,3-diketone-linked thymidine 5′-O-mono- and triphosphate have been synthesized via CuAAC click on response of diketone-alkynes with 5-azidomethyl-dUMP or -dUTP. The triphosphates have been good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5-dioxohexyl group (HDO) effectively reacted with arginine-containing peptides to type secure pyrimidine-linked conjugates, whereas the branched 2-acetyl-3-oxo-butyl (PDO) group was not reactive.
Response with Lys or terminal amino group shaped enamine adducts that have been vulnerable to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross-linking with Arg-containing peptides or proteins (e.g. histones).
Results of N-terminus modified Hx-amides on DNA binding affinity, sequence specificity, mobile uptake, and gene expression
5 X-HxIP (Hx-amides) 6a-e, during which the N-terminus p-anisyl moiety is modified, have been designed and synthesised with the aim of optimising DNA binding, bettering mobile uptake/nuclear penetration, and enhancing the modulation of the topoisomerase IIα (TOP2A) gene expression. The modifications embody a fluorophenyl group and different heterocycles bearing completely different molecular shapes, measurement, and polarity. Like their mother or father compound HxIP 3, all 5 X-HxIP analogues bind preferentially to their cognate sequence 5′-TACGAT-3′, which is discovered embedded on the 5′ flank of the inverted CCAAT box-2 (ICB2) web site within the TOP2A gene promoter, and inhibit protein advanced binding, as evidenced in a cell free system.
Curiously, the 4-pyridyl analog 6a displays higher binding affinity for the goal DNA sequence and abolishes the protein:ICB2 interplay in vitro, at a decrease focus, in comparison with the prototypical compound HxIP 3. Analogues 6b-e, show improved DNA sequence specificity, however lowered binding affinity for the cognate sequence, relative to the unmodified HxIP 3, with polyamides 6b and 6e being essentially the most sequence selective. Nonetheless, not like Three and 6b, 6a was unable to enter cells, entry the nucleus and thereby have an effect on TOP2A gene expression in confluent human lung most cancers cells.
These outcomes present that whereas DNA binding affinity and sequence selectivity are vital, consideration of mobile uptake and focus within the nucleus are important when exerting organic exercise is the specified final result. By characterising the DNA binding, mobile uptake and gene regulatory properties of those small molecules, we are able to elucidate the determinants of the elicited organic exercise, which might be impacted by even small structural modifications within the polyamide molecular design.
DNA and modified vaccinia Ankara prime-boost vaccination generates robust CD8 + T cell responses towards minor histocompatibility antigen HA-1
- Allogeneic immune responses underlie the graft-versus-leukaemia impact of stem cell transplantation, however illness relapse happens in lots of sufferers. Minor histocompatibility antigen (mHAg) peptides mediate alloreactive T cell responses and induce graft-versus-leukaemia responses when expressed on affected person haematopoietic tissue. We vaccinated 9 HA-1-negative donors towards HA-1 with a ‘prime-boost’ protocol of both two or three DNA ‘priming’ vaccinations previous to ‘increase’ with modified vaccinia Ankara (MVA).
- HA-1-specific CD8+ T cell responses have been noticed in seven donors with magnitude as much as 1·5% of whole CD8+ T cell repertoire. HA-1-specific responses peaked two weeks post-MVA problem and have been measurable in most donors after 12 months. HA-1-specific T cells demonstrated robust cytotoxic exercise and lysed goal cells with endogenous HA-1 protein expression.
- The sample of T cell receptor (TCR) utilization by HA-1-specific T cells revealed robust conservation of T cell receptor beta variable 7-9 (TRBV7-9) utilization between donors. These findings describe one of many strongest major peptide-specific CD8+ T cell responses but recorded to a DNA-MVA prime-boost routine and this may occasionally mirror the robust immunogenicity of mHAg peptides. Prime-boost vaccination in donors or sufferers could show of considerable profit in boosting graft-versus-leukaemia responses.
Conversion of RNA Aptamer into Modified DNA Aptamers Gives for Extended Stability and Enhanced Antitumor Exercise
Aptamers, artificial single-strand oligonucleotides which might be comparable in operate to antibodies, are promising as therapeutics due to their minimal unwanted effects. Nonetheless, the soundness and bioavailability of the aptamers pose a problem. We developed aptamers transformed from RNA aptamer to modified DNA aptamers that concentrate on phospho-AXL with improved stability and bioavailability. On the idea of the comparative evaluation of a library of 17 transformed modified DNA aptamers, we chosen aptamer candidates, GLB-G25 and GLB-A04, that exhibited the best bioavailability, stability, and strong antitumor impact in in vitro experiments.
Spine modifications similar to thiophosphate or dithiophosphate and a covalent modification of the 5′-end of the aptamer with polyethylene glycol optimized the pharmacokinetic properties, improved the soundness of the aptamers in vivo by lowering nuclease hydrolysis and renal clearance, and achieved excessive and sustained inhibition of AXL at a really low dose. Remedy with these modified aptamers in ovarian most cancers orthotopic mouse fashions considerably lowered tumor development and the variety of metastases.
This efficient silencing of the phospho-AXL goal thus demonstrated that aptamer specificity and bioavailability might be improved by the chemical modification of present aptamers for phospho-AXL. These outcomes lay the inspiration for the interpretation of those aptamer candidates and companion biomarkers to the clinic.
 100 BP DNA LADDER, 500UL PER KIT |
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| M-DNA-100BP | CORNING | 1/pk | EUR 73 |
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Description: Bioscience Mol Bio; DNA Ladder |
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 1 KB DNA LADDER, 500UL PER KIT |
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| M-DNA-1KB | CORNING | 1/pk | EUR 70 |
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Description: Bioscience Mol Bio; DNA Ladder |
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) Recombinant Shigella Flexneri trpR Protein (aa 2-108) |
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| VAng-Lsx08224-500gEcoli | Creative Biolabs | 500 µg (E. coli) | EUR 2071 |
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Description: Shigella Flexneri Trp operon repressor, recombinant protein. |
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Recombinant Shigella Flexneri trpR Protein (aa 2-108) |
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| VAng-Lsx08224-50gEcoli | Creative Biolabs | 50 µg (E. coli) | EUR 1425 |
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Description: Shigella Flexneri Trp operon repressor, recombinant protein. |
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) Recombinant Shigella Flexneri trpS Protein (aa 1-334) |
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| VAng-Lsx08225-1mgEcoli | Creative Biolabs | 1 mg (E. coli) | EUR 4600 |
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Description: Shigella Flexneri Tryptophan--tRNA ligase, recombinant protein. |
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Recombinant Shigella Flexneri trpS Protein (aa 1-334) |
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| VAng-Lsx08225-500gEcoli | Creative Biolabs | 500 µg (E. coli) | EUR 3060 |
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Description: Shigella Flexneri Tryptophan--tRNA ligase, recombinant protein. |
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Recombinant Shigella Flexneri trpS Protein (aa 1-334) |
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| VAng-Lsx08225-50gEcoli | Creative Biolabs | 50 µg (E. coli) | EUR 2099 |
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Description: Shigella Flexneri Tryptophan--tRNA ligase, recombinant protein. |
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) Recombinant Shigella Flexneri truA Protein (aa 1-270) |
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| VAng-Lsx08226-1mgEcoli | Creative Biolabs | 1 mg (E. coli) | EUR 3913 |
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Description: Shigella Flexneri tRNA pseudouridine synthase A, recombinant protein. |
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Recombinant Shigella Flexneri truA Protein (aa 1-270) |
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| VAng-Lsx08226-500gEcoli | Creative Biolabs | 500 µg (E. coli) | EUR 2773 |
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Description: Shigella Flexneri tRNA pseudouridine synthase A, recombinant protein. |
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Recombinant Shigella Flexneri truA Protein (aa 1-270) |
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| VAng-Lsx08226-50gEcoli | Creative Biolabs | 50 µg (E. coli) | EUR 1906 |
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Description: Shigella Flexneri tRNA pseudouridine synthase A, recombinant protein. |
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) Recombinant Shigella Flexneri truB Protein (aa 1-314) |
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| VAng-Lsx08227-1mgEcoli | Creative Biolabs | 1 mg (E. coli) | EUR 4463 |
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Description: Shigella Flexneri tRNA pseudouridine synthase B, recombinant protein. |
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Recombinant Shigella Flexneri truB Protein (aa 1-314) |
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| VAng-Lsx08227-500gEcoli | Creative Biolabs | 500 µg (E. coli) | EUR 2964 |
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Description: Shigella Flexneri tRNA pseudouridine synthase B, recombinant protein. |
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Recombinant Shigella Flexneri truB Protein (aa 1-314) |
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| VAng-Lsx08227-50gEcoli | Creative Biolabs | 50 µg (E. coli) | EUR 2030 |
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Description: Shigella Flexneri tRNA pseudouridine synthase B, recombinant protein. |
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) Recombinant Shigella Flexneri truD Protein (aa 1-349) |
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| VAng-Lsx08228-1mgEcoli | Creative Biolabs | 1 mg (E. coli) | EUR 4422 |
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Description: Shigella Flexneri tRNA pseudouridine synthase D, recombinant protein. |
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Recombinant Shigella Flexneri truD Protein (aa 1-349) |
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| VAng-Lsx08228-500gEcoli | Creative Biolabs | 500 µg (E. coli) | EUR 3129 |
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Description: Shigella Flexneri tRNA pseudouridine synthase D, recombinant protein. |
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Recombinant Shigella Flexneri truD Protein (aa 1-349) |
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| VAng-Lsx08228-50gEcoli | Creative Biolabs | 50 µg (E. coli) | EUR 2140 |
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Description: Shigella Flexneri tRNA pseudouridine synthase D, recombinant protein. |
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) Recombinant Shigella Flexneri tsf Protein (aa 2-283) |
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| VAng-Lsx08229-1mgEcoli | Creative Biolabs | 1 mg (E. coli) | EUR 3995 |
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Description: Shigella Flexneri Elongation factor Ts, recombinant protein. |
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Recombinant Shigella Flexneri tsf Protein (aa 2-283) |
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| VAng-Lsx08229-500gEcoli | Creative Biolabs | 500 µg (E. coli) | EUR 2828 |
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Description: Shigella Flexneri Elongation factor Ts, recombinant protein. |
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Recombinant Shigella Flexneri tsf Protein (aa 2-283) |
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| VAng-Lsx08229-50gEcoli | Creative Biolabs | 50 µg (E. coli) | EUR 1934 |
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Description: Shigella Flexneri Elongation factor Ts, recombinant protein. |
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) Recombinant Shigella Flexneri tsgA Protein (aa 1-393) |
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| VAng-Lsx08230-1mgEcoli | Creative Biolabs | 1 mg (E. coli) | EUR 5040 |
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Description: Shigella Flexneri Protein tsgA, recombinant protein. |
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Recombinant Shigella Flexneri tsgA Protein (aa 1-393) |
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| VAng-Lsx08230-1mgYeast | Creative Biolabs | 1 mg (Yeast) | EUR 6305 |
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Description: Shigella Flexneri Protein tsgA, recombinant protein. |
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) Recombinant Shigella Flexneri tufA Protein (aa 2-394) |
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| VAng-Lsx08232-1mgEcoli | Creative Biolabs | 1 mg (E. coli) | EUR 4999 |
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Description: Shigella Flexneri Elongation factor Tu, recombinant protein. |
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Recombinant Shigella Flexneri tufA Protein (aa 2-394) |
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| VAng-Lsx08232-500gEcoli | Creative Biolabs | 500 µg (E. coli) | EUR 3322 |
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Description: Shigella Flexneri Elongation factor Tu, recombinant protein. |
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Recombinant Shigella Flexneri tufA Protein (aa 2-394) |
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| VAng-Lsx08232-50gEcoli | Creative Biolabs | 50 µg (E. coli) | EUR 2264 |
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Description: Shigella Flexneri Elongation factor Tu, recombinant protein. |
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) Recombinant Shigella Flexneri ubiA Protein (aa 1-290) |
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| VAng-Lsx08233-inquire | Creative Biolabs | inquire | Ask for price |
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Description: Shigella Flexneri 4-hydroxybenzoate octaprenyltransferase, recombinant protein. |
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) Recombinant Shigella Flexneri ubiB Protein (aa 1-546) |
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| VAng-Lsx08234-inquire | Creative Biolabs | inquire | Ask for price |
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Description: Shigella Flexneri Probable ubiquinone biosynthesis protein UbiB, recombinant protein. |
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) Recombinant Shigella Flexneri ubiC Protein (aa 1-165) |
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| VAng-Lsx08235-1mgEcoli | Creative Biolabs | 1 mg (E. coli) | EUR 3459 |
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Description: Shigella Flexneri Chorismate--pyruvate lyase, recombinant protein. |
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Recombinant Shigella Flexneri ubiC Protein (aa 1-165) |
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| VAng-Lsx08235-500gEcoli | Creative Biolabs | 500 µg (E. coli) | EUR 2333 |
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Description: Shigella Flexneri Chorismate--pyruvate lyase, recombinant protein. |
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Recombinant Shigella Flexneri ubiC Protein (aa 1-165) |
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| VAng-Lsx08235-50gEcoli | Creative Biolabs | 50 µg (E. coli) | EUR 1604 |
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Description: Shigella Flexneri Chorismate--pyruvate lyase, recombinant protein. |
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) Recombinant Shigella Flexneri uhpA Protein (aa 1-196) |
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| VAng-Lsx08236-1mgEcoli | Creative Biolabs | 1 mg (E. coli) | EUR 3445 |
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Description: Shigella Flexneri Transcriptional regulatory protein uhpA, recombinant protein. |
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Recombinant Shigella Flexneri uhpA Protein (aa 1-196) |
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| VAng-Lsx08236-500gEcoli | Creative Biolabs | 500 µg (E. coli) | EUR 2470 |
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Description: Shigella Flexneri Transcriptional regulatory protein uhpA, recombinant protein. |
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Recombinant Shigella Flexneri uhpA Protein (aa 1-196) |
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| VAng-Lsx08236-50gEcoli | Creative Biolabs | 50 µg (E. coli) | EUR 1673 |
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Description: Shigella Flexneri Transcriptional regulatory protein uhpA, recombinant protein. |
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) Recombinant Shigella Flexneri ulaR Protein (aa 1-251) |
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| VAng-Lsx08237-1mgEcoli | Creative Biolabs | 1 mg (E. coli) | EUR 3789 |
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Description: Shigella Flexneri HTH-type transcriptional regulator UlaR, recombinant protein. |
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Recombinant Shigella Flexneri ulaR Protein (aa 1-251) |
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| VAng-Lsx08237-500gEcoli | Creative Biolabs | 500 µg (E. coli) | EUR 2718 |
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Description: Shigella Flexneri HTH-type transcriptional regulator UlaR, recombinant protein. |
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Recombinant Shigella Flexneri ulaR Protein (aa 1-251) |
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| VAng-Lsx08237-50gEcoli | Creative Biolabs | 50 µg (E. coli) | EUR 1838 |
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Description: Shigella Flexneri HTH-type transcriptional regulator UlaR, recombinant protein. |
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) Recombinant Shigella Flexneri umuD Protein (aa 1-139) |
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| VAng-Lsx08238-1mgEcoli | Creative Biolabs | 1 mg (E. coli) | EUR 3088 |
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Description: Shigella Flexneri Protein umuD, recombinant protein. |
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Recombinant Shigella Flexneri umuD Protein (aa 1-139) |
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| VAng-Lsx08238-500gEcoli | Creative Biolabs | 500 µg (E. coli) | EUR 2209 |
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Description: Shigella Flexneri Protein umuD, recombinant protein. |
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Recombinant Shigella Flexneri umuD Protein (aa 1-139) |
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| VAng-Lsx08238-50gEcoli | Creative Biolabs | 50 µg (E. coli) | EUR 1521 |
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Description: Shigella Flexneri Protein umuD, recombinant protein. |
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