Mgbe akwụkwọ na-ezo aka Vectors Vendor: Otu esi agụ amaokwu Reagent (Ọmụmụ ihe: GenScript pcDNA3 na nyocha MDM2)
Mgbe ndị isi nyocha na ndị ọkà mmụta sayensị dị elu na-enyocha akwụkwọ ndị e bipụtara ọhụrụ na oncology ma ọ bụ usoro ihe ọmụmụ cell., A na-ewerekarị akwụkwọ ndị na-ere ahịa dị ka stampụ nkwado doro anya. Ịhụ vector ma ọ bụ peptide omenala a kpọtụrụ aha n'ofe akwụkwọ iri abụọ ndị ọgbọ nyochara na-emepụta echiche nke ntụkwasị obi.. Ọ bụrụ na ọtụtụ ụlọ nyocha nọọrọ onwe ha na-azụta otu GenScript pcDNA3 vector construct ma ọ bụ peptide sịntetik azụmahịa iji mụọ akara MDM2-p53, ọ dị ka ihe ezi uche dị na ya ịtụ otu nọmba katalọgụ ahụ ma gaba n'ihu ozugbo na ogbugbu bench.
Agbanyeghị, emeso ndị na-ere ahịa dị ka onye nnọchi anya maka ịdịmma batch pụtara ìhè bụ otu n'ime ihe na-ebutekarị data ụlọ nyocha na-enweghị atụ.. Ntụtu aka na akwụkwọ akụkọ ndị ọgbọ nyochara na-edepụta ojiji akụkọ ihe mere eme, ọ bụghị ọtụtụ-kpọmkwem anụ ahụ, kemịkalụ, ma ọ bụ nkwenye ọrụ. Ọkpụkpụ plasmid azụmaahịa na-aga n'usoro nlegharị anya n'usoro nke ndị na-ere ahịa, ngafe n'ụdị ndị ọbịa na-ewebata obere ọnụọgụ dị iche iche, na vectors nke ndị na-akwalite nje na-akwali nwere ike ịkpa àgwà na-atụghị anya ya dabere na mkpụrụ ndụ ihe nketa cellular..
Ichekwa mmụgharị nnwale chọrọ ịmụta ka esi agụ ngụ akwụkwọ nke ọma na ime onye nwere onwe ya., njikwa ogo orthogonal (QC) protocol tupu ịnakwere ọ bụla akwụkwọ-eji reagent.
Nkọwa ndị na-ere ahịa azụmaahịa vs. Nkwado mmemme: Na-emebi akara ngosi ntụkwasị obi na-apụtaghị ìhè
Iji nyochaa ihe ndị sitere n'akwụkwọ nke ọma, ụlọ nyocha ga-amata ọdịiche dị n'etiti akara azụmahịa dị iche iche. Na mbipụta sayensị, ere kwuru, ihe ndekọ data, na nkwado mmemme na-arụ ọrụ nhazi na nkwado dị iche iche, ma ọ dịghị nke ọ bụla n'ime ha na-ekwe nkwa na a ọhụrụ otutu si a azụmahịa soplaya ga-arụ ọrụ identically na gị assay.
Aha akwụkwọ / RRID → Ndekọ ojiji akụkọ ihe mere eme (Ebe a zụrụ ya)
Nkwado onye na-ere ahịa → Mgbama ahịa azụmaahịa (Visibiliti ika)
Orthogonal QC & CoA → Nnwale ihe omimi (Njiri mara ọtụtụ & ịdị ọcha)
Ngụ akwụkwọ na ihe nchọpụta akụrụngwa nyocha (RRIDs)
Mgbe mpempe akwụkwọ depụtara nọmba katalọgụ ma ọ bụ ihe nchọpụta akụrụngwa nyocha (ACHỌRỌ M), ọ na-ewepụta traceability maka data ebipụtara. Omume nhota ahaziri ahazi na-akwalite nkwenye na mmụgharị ihe n'ezie site n'ikwe ka ndị na-agụ akwụkwọ soro usoro akụrụngwa n'ofe ọmụmụ., dị ka pụta ìhè na Ntuziaka mbipụta NIH maka nkwenye na ngụpụta data.
Ngwa ikpo okwu na-egwuputa ihe dị ka CiteAb na-achịkọta nkọwa ndị a iji tụọ nkuchi ahịa na usoro ojiji., na-enye nghọta bara uru na ọnụnọ ndị na-ere ahịa, dị ka akọwapụtara n'ime Nyocha ngụpụta ngwaahịa sayensị ndụ CiteAb. Na-ehota kpọmkwem nọmba katalọgụ ngwaahịa, ọtụtụ ọnụọgụ, na RRID na-ahụ na ndị ọgbọ sayensị nwere ike ịchọpụta ihe dị n'okpuru, dị ka akọwara na Ntuziaka StressMarq na ngụ ngwaahịa RRID.
Agbanyeghị, ngụ akwụkwọ na-edekọ ihe otu ndị nyocha gara aga zụtara-ọ naghị akwado na reagent ahụ kwadoro onwe ya tupu ebipụta nnwale ha..
Ntugharị igodo: Ntụle ndị ọgbọ nyochara na-akwado na ejiri otu akụrụngwa onye na-ere ahịa mee ihe n'ọmụmụ ihe e bipụtara, mana ọ naghị edochi anya anụ ahụ akọwapụtara nke ukwuu, usoro, ma ọ bụ nkwenye na-arụ ọrụ na ụlọ nyocha nke gị.
Nkwado Mmemme Azụmahịa na Nhụta Akara
Nkwado mmemme—dị ka ụlọ ntu ndị na-ere ahịa na nnọkọ isi okwu, ulo oru nka akwadoro, ma ọ bụ enyemaka njem sayensị - na-arụ ọrụ naanị dị ka akara ngosi ọhụụ azụmahịa. Nkwado na-egosipụta ike ahịa nke onye na-ere ahịa, ọ bụghị nyocha ịdị ọcha ma ọ bụ ntụkwasị obi arụ ọrụ nke reagents ndu ha.
Ịdabere na ama ama ama ama ama ma ọ bụ ngụ akwụkwọ agụmagụ ugboro ugboro dị ka onye nọchiri anya njikwa mma dị n'ime na-emepụta nnukwu ihe egwu dị ndụ., karịsịa n'ụzọ usoro nhazi mmetụta dị ka MDM2/p53 mgbaàmà.
Ọmụmụ ihe: Ewupụta nkwupụta okwu GenScript pcDNA3 na nyocha akara ngosi MDM2/p53
Iji ghọta ka vector ndị e zoro aka n'akwụkwọ nwere ike isi webata mgbanwe nnwale, tulee iji GenScript pcDNA3 okwu na-ewu na nyocha MDM2.
Òké nkeji abụọ 2 (MDM2) bụ E3 ubiquitin ligase nke na-arụ ọrụ dị ka onye na-achịkwa ihe na-adịghị mma nke protein p53 tumor suppressor.. N'ime ụdị anụ ọhịa p53 sistemụ, MDM2 na-ejikọta ngalaba ngbanwe N-terminal nke p53, ezubere ya maka mbibi proteasomal na akagide nzaghachi nzaghachi adịghị mma, dị ka nkọwa na nyocha zuru oke na imepụta ọgwụ ugbu a. N'ihi na MDM2 overexpression nwere ike ịkwụsị ọrụ p53 na-enweghị mmụgharị mkpụrụ ndụ, Ndị nchọpụta na-ebugharị plasmids MDM2 recombinant ugboro ugboro iji nyochaa njide okirikiri cell, apoptosis, ma ọ bụ obere-molecule MDM2-p53 inhibitors.
pcDNA3 Okwu Vector Backbone CMV Promoter → Epitope Tag → MDM2 Ntinye → BGH PolyA (P53 kwadoro ya) (Nwere ike ịgbanwe E3) (Ngalaba mgbanaka) (Nkwụsi ike transcript)
Plasmids dị ka pcDNA3 MDM2 WT ka edekọtara na ebe nchekwa agụmakwụkwọ, gụnyere Addgene pcDNA3 MDM2 WT nchekwa data, ma megharịa ma ọ bụ gbanwee site na ndị na-ere ahịa dịka GenScript. Ọkpụkpụ azụ vector pcDNA3 (5.4 kb) A na-eji cytomegalovirus mmadụ (CMV) ngwa ngwa-nkwalite ngwa ngwa maka ọkwa anụ mammalian dị elu, mmalite SV40 maka mmegharị episomal na SV40 nnukwu T-antigen na-egosipụta sel ndị ọbịa (dị ka HEK293T), yana akara nhọpụta neomycin/G418.
Nleba nka 1: Mmegide CMV na-akwalite site na ụdị anụ ọhịa p53
Nnukwu ọnyà dị na ọmụmụ okwu MDM2 gụnyere iwu nkwalite. Ọ bụ ezie na a na-emeso onye na-akwalite CMV na pcDNA3 dị ka onye nkwado siri ike, Ihe omumu ihe omumu gosiputara na a na-egbochi oru onye na-akwalite CMV site na ọkwa p53 di elu di elu., dị ka e gosiri na nnyocha e bipụtara na Journal of Biological Chemistry. Rasa Research Peptides Supplier
Mgbe ndị nyocha na-ebufe pcDNA3-MDM2 n'ime sel nwere akara p53 na-emebibeghị, ọkwa p53 na-ebili na-egbochi onye nkwalite CMV, na-akpata na-abụghị linear, okwu MDM2 na-amachi onwe ya. Ọ bụrụ na otu nchọcha na-ewere ọrụ nkwalite mgbe niile dabere na ngụpụta akwụkwọ, enwere ike ịkenye mgbanwe na ụba protein MDM2 iji nwalee nrụpụta ngwa ngwa ma ọ bụ mmebi nke ntụgharị asụsụ karịa nkwubi ndị nkwalite..
Ihe ndekọ oche oche (MOL na-agbanwe ụlọ nyocha njikwa ogo): N'ime oge ọ bụla n'ime nkwado ndabere nke akwụkwọ pcDNA3 plasmid sitere na akwụkwọ, usoro nke Sanger bidirectional gafee njikọ ntinye ihe nkwalite achọpụtara ụdị dị iche iche nke otu nucleotide enweghị nkewa. (SNV) n'ime CMV ozugbo-n'oge nkwalite mpaghara. Ntugharị obere usoro ndị a butere mbelata ~ 35% na ọrụ ndị na-akwalite ntọala n'ụdị cell ụdị anụ ọhịa ma e jiri ya tụnyere maapụ ntụaka NCBI nke canonical — na-egosipụta ihe kpatara nkwenye usoro mkpụrụ ndụ ihe nketa ji bụrụ iwu tupu emee nyocha cellular ala..
Nleba nka 2: Nkpado Epitope na Mgbanwe ngalaba MAKA-Mkpịsị aka
Iji soro recombinant MDM2, vectors azụmahịa na-etinyekarị mkpado N-terminal ma ọ bụ C-terminal epitope (dị ka Myc, Ọkọlọtọ, ma ọ bụ GFP). MDM2 na-adabere na ngalaba mkpịsị aka C-terminal RING maka ọrụ E3 ubiquitin ligase na homodimerization.. Peptides nwere stapled
Ọ bụrụ na onye na-ere ahịa na-agbanwe njikọ nke cloning ma ọ bụ ọ bụrụ na etinyere mkpado n'akụkụ ngalaba mkpịsị aka RING na-enweghị nkwado siri ike, protein fusion egosipụtara nwere ike igosipụta ọrụ E3 ligase belatara ma ọ bụ nkọwapụta mkpụrụ agbanwe agbanwe. Usoro nke ntinye ntinye na orthogonal mass spectrometry dị mkpa iji gosi na mkpado ahụ emebibeghị mpịakọta ngalaba na-arụ ọrụ..
Ihe ize ndụ zoro ezo nke ndị na-eji akwụkwọ eme ihe: Usoro Drift, Mkpado nnyonye anya, na Batch Variation
Ịnabata akwụkwọ e zoro aka na reagents na-enweghị nkwenye na-ewebata ụdị ụzọ ọdịda atọ dị mkpa:
- Mgbanwe na-enweghị nkewa na usoro Drift: Plasmids na-agbasa n'ofe nje nje bacteria nwere ike ịkpakọta mmụkọ ihe, ntinye, ma ọ bụ transposon ọcha. Ịgafe plasmid preps na-enweghị usoro ntinye ókèala nwere ike gbanwee ike onye na-akwalite ma ọ bụ webata mmụgharị dị jụụ / enweghị isi na etiti koodu..
- Peptide na ihe ndị na-adịghị ọcha reagent: Na njikọ nke peptide (dị ka MDM2-binding helical peptides ma ọ bụ p53-enwetara tracer peptides.), ndị na-ebubata azụmahịa nwere ike ibuga ihe nwere usoro nhichapụ mpịachi ((n-1) ma ọ bụ (n-2) isomers) ọ bụrụ na ịdị ọcha ezughị oke. peptides sịntetik na-adịghị ọcha nwere ike ime ka kinetics na-ejikọta nke ọma na resonance plasmon. (SPR) ma ọ bụ fluorescence polarization (FP) nyocha.
- Esemokwu CoA na enweghị ntụkwasị obi Lot: Asambodo nyocha (CoAs) nke ndị na-ebubata ya nyere nwere ike ịnọchite anya nnwale batch akụkọ ihe mere eme karịa vial akọwapụtara na ụlọ nyocha gị. Na-akwadoghị nzaba traceability na nwale nkwụsi ike nchekwa, Ọdịiche dị ọcha nke otutu-na-ọza nwere ike imebi mmụgharị ọnụọgụgụ.
Maka ndụmọdụ: Echela na Asambodo Analysis nke azụmahịa na-egosipụta ọnọdụ anụ ahụ dị ugbu a nke reagent mgbe njem gafere. Na-eme nyocha nke ime na nyocha ịdị ọcha mgbe niile mgbe nnata gasịrị.
Protocol nkwado nnwere onwe nke 3 maka ndị reagents sitere na akwụkwọ
Iji chebe ọrụ nyocha site na arịa reagenti enwetaghị nkwenye, ụlọ nyocha kwesịrị ime ka a haziri ahazi, usoro nkwado orthogonal atọ nke ọkwa atọ.
Ogbo 1: Nyocha usoro ọmụmụ → Ogbo 2: Usoro & QC anụ ahụ → Ogbo 3: Njikwa arụ ọrụ
(CoA & Achọrọ m ịlele) (Onye egwu/NGS & RP-HPLC/MS) (p53 - efu & Nyocha nke Proteasome)
Ogbo 1: Nyocha nke usoro ọmụmụ na otutu traceability
Tupu iwepu ma ọ bụ bufee vector ọhụrụ ma ọ bụ peptide sịntetịt, nyochaa akwụkwọ ya zuru oke:
- Dekọọ metadata ihe: Detuo aha onye na-ere ihe, kpọmkwem katalọgụ nọmba, NJ clone, batch / otutu nọmba, na RRID.
- Nyochaa Map Vendor na CoA: Tulee maapụ plasmid onye na-ere ahịa wetara ma ọ bụ
Nke Peptides Mepụta Ahịrị friza: Maka plasmid, soro ndị ọbịa nje nje, nchịkọta nhọrọ ọgwụ nje, na nọmba ngafe sitere na mgbanwe mbụ.
nchịkọta nhọrọ ọgwụ nje, na nọmba ngafe sitere na mgbanwe mbụ.
Ogbo 2: Usoro Orthogonal na njirimara anụ ahụ
A ga-emerịrị nkwado anụ ahụ na mkpụrụ ndụ ihe nketa tupu ịme nyocha nke ndu:
- Usoro nke Sanger: Mee usoro Sanger bidirectional n'ofe njikọ ntinye nkwalite niile, mkpado epitope, na saịtị mmụpụta arụrụ arụ.
- Usoro nke ọgbọ na-abịa (NGS): Maka mgbagwoju anya, ogologo, ma ọ bụ multicistronic vectors, jiri usoro ịgụ ogologo oge (dị ka Oxford Nanopore ma ọ bụ PacBio) iji chọpụta ụdịdị obere ugboro ma ọ bụ nhazi azụ azụ nke usoro Sanger nwere ike ịtufu..
- RP-HPLC na Mass Spectrometry (MS) maka Peptides: Mgbe ị na-eji synthetic peptide reagents, nyochaa ihe atụ site na iji nyocha C18 Reverse-Phase HPLC (0.1% TFA na H₂O/ACN gradient) iji hụ na mkpebi kacha elu (Rₛ ≥ 1.5) na ịdị ọcha (≥ 98%). Gosipụta ịdị arọ molekụla site na iji Electrospray Ionization Mass Spectrometry dị elu (ESI-MS) na a uka njehie n'okpuru 5 ppm.
Maka ụlọ nyocha na-achọ njikọ omenala na nkwenye a pụrụ ịdabere na ya, eji a omenala peptide njikọ ikpo okwu kwadoro site keukwu HPLC nyocha na ọrụ nnwale spectrometry na-achọpụta na usoro ọ bụla ewepụtara na-enweta njirimara siri ike tupu eji ya.
Ogbo 3: Njikwa ụzọ ụzọ na-arụ ọrụ
Nkwenye ihe ndị dị ndụ na-akwado na protein egosipụtara ma ọ bụ peptide arụpụtara na-arụ ọrụ nke ọma na ọnọdụ cellular ebumnuche:
- Ngosipụta nkwupụta na nha: Mee nyocha nke Western blot ma ọ bụ immunoprecipitation nyocha iji gosi na nrụpụta ahụ egosipụtara dabara na ịdị arọ nke molekụla buru amụma na-enweghị ngwaahịa mmebi na-atụghị anya ya..
- Njikwa okirikiri mkpụrụ ndụ ihe nketa cellular: Mgbe ị na-enyocha ihe nrụpụta MDM2, gụnyere ahịrị cell p53-null (dị ka Saos-2 ma ọ bụ H1299) n'akụkụ p53-wildtype ahịrị (dị ka MCF-7 ma ọ bụ U2OS) ịmata ọdịiche dị n'ihe nsi na-adabere p53 na mmetụta vector na-apụ apụ.
- Nyocha Mgbochi Proteasome: Jiri ihe mgbochi proteasome na-agwọ sel ndị ebufere (dị ka MG132) iji gosi na enwere ike ịzọpụta mbibi p53 na-agbasa MDM2, guzobe ọrụ E3 ligase na-arụ ọrụ.
Framework tabular: Ndepụta ụlọ nyocha maka nkwado reagent na mmụgharị
Ndepụta nleba anya na-esonụ na-enye usoro ahaziri ahazi maka
Nkwado Ụlọ ọrụ Peptide a pụrụ ịdabere na ya Ogbo
zoro aka na vectors na omenala peptide reagents tupu emegbu nnwale:
| Ọkwa nkwado | Oke / Nwalee | Usoro nnabata | Mgbochirị ihe arụrụ arụ |
|---|---|---|---|
| Ogbo 1: Usoro ọmụmụ | Ọtụtụ traceability & Nyochaa CoA | Ọnụọgụ batch dakọtara
Onye nrụpụta Liraglutide Api 100% njirimara usoro gafee mgbasa ozi, mkpado, ma tinye oke > Ịnweta isoforms plasmid ezighi ezi ma ọ bụ ihe katalọgụ gbanwere |
|
| Ogbo 2: Usoro | Sanger / Usoro NGS Junction | 100% njirimara usoro gafee mgbasa ozi, mkpado, ma tinye oke | Mmụba ngbanwe nke etiti, mmụkọ ọnụ, na nkwụsị nkwụsị |
| Ogbo 2: QC nke anụ ahụ | Nyocha Ọcha RP-HPLC | Otu ọnụ ọnụ chromatographic dị nkọ, ịdị ọcha ≥ 98%, Rₛ ≥ 1.5 | Usoro mkpochapụ agbajiela (n-1) na njikọ akụkụ-ngwaahịa |
| Ogbo 2: Ọnụ ego QC | ESI-MS nwere mkpebi dị elu Peptide Synthesis | Monoisotopic mass izi ezi n'ime < 5 ppm nke theoretical uka | Ndochi amino acid na-ezighi ezi ma ọ bụ ihe nchebe na-ezughị ezu |
| Ogbo 3: Na-arụ ọrụ | Western Blot & Nkwenye nkwupụta | Ngwakọta arọ molekụla ziri ezi; ọ dịghị na-atụghị anya cleavage ngwaahịa | Protein na-atụgharị uche, nbibi ngwa ngwa, ma ọ bụ nkwuputa okwu dara jụụ |
| Ogbo 3: Ụzọ ụzọ | Isogenic & Njikwa Cell efu | Kpochapụ ọdịiche ọrụ na p53-wildtype vs. p53 - mkpụrụ ndụ efu | Ngosipụta ụgha nke vector toxicity na akara ụzọ a kapịrị ọnụ |
Chekwaa mmeghari nnwale na Vector omenala na Peptide R&D
Ngụ akwụkwọ na-enye mmalite maka nhọrọ reagent, mana ha enweghị ike dochie anya njikwa mma n'ime. Dị ka nyocha biomedical na-eche ihu nyocha na-eto eto maka nrụpụta data, Ụlọ nyocha nke na-akwado onwe ya na vector ọ bụla na-arụ na peptide batch na-echebe ego ha na-etinye na bench, gbochie awa nyocha efu, ma hụ na nkwubi okwu ebipụtara ka siri ike.
Mgbe ị na-enweta ihe pụrụ iche ma ọ bụ na-ebuli usoro omenala, na-arụkọ ọrụ na onye mmekọ nyocha nke na-arụ ọrụ n'ime Klas 100 akụrụngwa ime ụlọ dị ọcha na-eme ka o doo anya na a na-ewepụ mmetọ cross na gburugburu ebe obibi endotoxins na mmepụta. Ọzọkwa, itinye aka pụrụ iche peptide CRO ngwọta na-enye ohere ka ndị otu nyocha na-enweta ihe edere n'ụzọ zuru ezu-nke chromatograms HPLC n'otu n'otu na-akwado, ESI-MS ụdịdị, na otutu-kpọmkwem CoAs—tupu ibido nyocha mkpụrụ ndụ dị egwu.
Site na ijikọ nyocha agụmagụ dị oke egwu na usoro nkwado siri ike nke ọkwa atọ, ụlọ nyocha nyocha nwere ike iji obi ike were ngwaọrụ ndị edepụtara n'akwụkwọ ma na-echekwa iguzosi ike n'ezi ihe na mmeghari nke mmepụta sayensị ha..
Nzọụkwụ ọzọ maka R&D Otu
Chọrọ njirimara onwe ma ọ bụ njikọ peptide omenala dị elu maka pipeline nyocha gị? Kpọtụrụ ndị ọkachamara nyocha na MOL Mgbanwe iji rịọ maka nyocha teknụzụ, nyochaa usoro nyocha nyocha, ma ọ bụ nyochaa usoro nhazi ahaziri ahazi dabere na ihe nnwale gị.
Banyere ndị nyocha sayensị
Ndị ọrụ dere ma nyochaa ntuziaka nka MOL na-agbanwe bọọdụ ndụmọdụ sayensị & Otu nyocha- otu jikọtara ndị agadi peptide chemists, ndị ọkà mmụta ihe ọmụmụ molecular, na nyocha QC ọkachamara. Na-arụ ọrụ na klas 100 akụrụngwa ime ụlọ dị ọcha, otu MOL Changes ọkachamara na nhazi usoro peptide omenala, microbial/njikọ siri ike, yana nkwado nyocha siri ike nke ukwuu (RP-HPLC, ESI-MS, na ule ịmụ nwa) iji kwado nchọpụta sayensị nwere ike imegharị.
