Kedu ihe kpatara nyocha Protein-Degradation chọrọ Peptide ka mma & Protein Reagent

Kedu ihe Reagent na-enyocha mbibi ga-eme n'ezie

Tupu nyochaa QC, ọ na-enyere aka ikewa klaasị reagent metụtara. Ntuziaka proteomic na kemịkalụ ọkọlọtọ na-ekewa ndị nta akụkọ fluorescent, nyocha dabere na ọrụ, na mmekọrịta-enrichment reagents, nke ọ bụla na-agbanwe ubiquitin-proteasome usoro (UPS) arụ ọrụ n'ime ihe agụpụta nwere ike ịtụ n'ime mkpụrụ ndụ, lysates, ma ọ bụ mmeghachi omume na-enweghị cell.

• Ihe akara fluorescent ka ị na-ahụ anya ma ọ bụ chọpụta enzymes na-arụ ọrụ, akụkụ nke proteasome, ma ọ bụ onye nta akụkọ sitere na in-gel fluorescence, microscope, ma ọ bụ ndị nta akụkọ nweta/ofu nke mgbaàmà.

  

• Akara aha biotin bụ ngwá ọrụ ịba ọgaranya, na-ejide deubiquitinases na-arụ ọrụ (DUB) ma ọ bụ protein gbanwetụrụ site na streptavidin kwaturu tupu immunoblotting ma ọ bụ mass-spectrometry njirimara.

• Ubiquitin na NEDD8-mimetic nyocha na fluorogenic ma ọ bụ FRET substrates na-ajụ enzymes na ligases nke ga-amata ọdịiche dị na ubiquitin site na ndị na-emegharị ubiquitin - kemịkalụ na-ekpebi ma ihe omume mbibi ezubere iche mere n'ezie..

  

• Ihe ndị na-eweda ala na-ebu usoro na-emebi emebi ma ọ bụ detron ka mfu na-akpata ma ọ bụ nke na-adabere na proteasome wepụta mgbanwe mgbaama enwere ike..

Nke ọ bụla n'ime ọrụ ndị a na-azụ ahịa na obere nkọwa nhazi: nke fọdụrụ ka akpọrọ, ebe mgbanwe na-anọdụ ala, ma njikọ isopeptide ma ọ bụ oxyester ziri ezi, na ma onye isi agha nọ na njedebe ziri ezi. Reagent nke “95% ịdị ọcha” na-ezochi ihe na-adịghị mma ma ọ bụ ihichapụ isomer nwere ike ịgbanwe njikọ., nkwụsi ike, ma ọ bụ ọnụego nbibi pụtara nke ọma iji megharịa nkọwa nke ihuenyo dum.

Peptides akpọpụtara chọrọ nkwenye akọwapụtara kpọmkwem

Mkpado fluorescent ma ọ bụ biotin abụghị ihe mgbakwunye na-agafe agafe. Njikọta na-agbanwe ma oge njide yana ionization na chromatography, na ọ na-ewebata a amụma uka mgbanwe - ma naanị ma ọ bụrụ na e bu n'obi umu bụ ihe kpụrụ.

Ogo reagent ebe a pụtara ikwado akpọrọ ụdị, ọ bụghị naanị peptide nne na nna. Nke ahụ na-achọkarị:

• Peptide Synthesis Nyochaa oke-spectrometry nke akara agbakwunyere site na ngbanwe oke a na-atụ anya ya;

• LC-MS ma ọ bụ UPLC na-enyocha iji gosi na molecule akpọrọ elutes na ionizes dị ka otu akọwapụtara., ụdị kachasị;

• mkpesa njirimara dị iche maka akara, n'ihi na omume ya gbanwere nwere ike kpuchie ngwaahịa ndị na-emekọ ihe ọnụ.

Maka biotin, ihe na-echegbu bụ ikwado ntụkwasị obi: Otu akụkụ nke akpọrọ ma ọ bụ mis-biotinylated nwere ike jikọta streptavidin n'ụzọ dị iche ma wetuo usoro protein na-ezighi ezi.. Maka fluorophores, Ụdị ọdịda ahụ dị iche mana ọ na-akpaghasịkwa ya - adịghị ọcha nke na-eme ka ọ bụrụ ogologo oge nyocha nke na-egosipụta ezigbo akara ma na-ebuli ọnụ ahịa.. Ụlọ nyocha nyocha na-ajụkarị ndị na-ere ahịa ka ha kọọ kpọmkwem oke akpọrọ na chromatogram nke njikọ ikpeazụ., ọ bụghị naanị usoro ịdị ọcha.

A ga-enyocharịrị mgbanwe mgbanwe assay-kpọmkwem, Echeghị

Ihe reagents na-eweda ala anaghị abụkarị peptides nkịtị. Ha na-ebu ubiquitin-mimetic ma ọ bụ NEDD8-mimetic ọcha, electrophilic warheads, njikọ aka, njikọ, na FRET ma ọ bụ ụzọ abụọ fluorogenic. Ibu akaebe dị na nkwenye na bu n'obi Ọdịdị na-achịkwa nza na na ọ dịghị isomer na-atụghị anya ya, nkwụsịtụ, ma ọ bụ ngwaahịa dị n'akụkụ akwakọbagoro n'ọkwa nke na-akpaghasị nyocha ahụ.

N'ihi na obere-molekul akụkụ nke degraders, omume nyocha apụtalarị nke ọma: ngwugwu siri ike na-eji LC-MS ma ọ bụ HPLC maka ịdị ọcha organic, NMR ma ọ bụ qNMR maka nhazi na nguzozi oke, na mgbe mgbe nyocha nke chiral ma ọ bụ ígwè iji jide stereocenter epimerization na trace-metal conteminants nke nwere ike imenyụ nhazi zinc.. The peptide na protein reagents na-eri nri otu ihuenyo kwesịrị otu ịdọ aka ná ntị - nkwenye nke mgbanwe saịtị, nkwenye nke njikọ na njedebe, ma dokwuo anya ma adịghị ọcha na-achị achị nwere ike igbochi ọgụgụ ihe ndu ndu.

Usoro Ikwesị ntụkwasị obi bụ enweghị nkwekọrịta

A na-enyekarị iwu ụlọ ọba akwụkwọ nyocha na usoro nyocha megide usoro akọwapụtara nke ọma nke na-etinye koodu degron, ihe njiri mara, ebe igbutu enzyme, ma ọ bụ mgbama dabere na phosphorylation. Ikwesị ntụkwasị obi nke usoro pụtara na nhazi ahụ bụ ihe ebupu n'ezie - na-enweghị ihichapụ, enweghị nnọchi, enweghị mgbaghara, ma kwụ ọtọ N- na C-termini.

Ikwesị ntụkwasị obi bụ ebe mkpirisi ọkwa ọkwa nyocha na-apụtakarị. Onye na-ere ahịa na-akwado oke oke naanị nwere ike tufuo isomer nhichapụ nwere otu oke aha., ma ọ bụ saịtị phospho arụnyere na ihe fọdụrụ na-ezighi ezi. Ntụkwasị obi n'ọkwa usoro chọrọ maapụ peptide ma ọ bụ nkewa MS/MS, amino acid analysis maka mejupụtara, na - maka ihe arụrụ arụ nke ukwuu - nkwenye na otu ọ bụla arụnyere na-anọdụ ala na saịtị echere. Mgbe ihuenyo mmejọ dara ada ma ọ bụ nsonaazụ DUB na-agbagwoju anya laghachiri na "usoro mkpụrụ osisi ahụ ezighi ezi site na otu ihe fọdụrụ.,” ọnụ ahịa ahụ bụ izu nke data enweghị ike imepụta nke ọ nweghị sọftụwia ala nwere ike idozi.

Nkwenye nyocha Orthogonal na-ebuli ntụkwasị obi

Otu mgbaama nyocha adịkarịghị edozi njirimara. Ntuziaka European Medicine Agency na nke mmepe Peptides sịntetik na imepụta peptides sịntetik na-atụ aro ma ọ dịkarịa ala ụzọ abụọ orthogonal maka njirimara, na orthogonal size-, ụgwọ-, na ụzọ dabere na hydrophobicity maka njirimara ịdị ọcha.

Nkwenye nke orthogonal dị mkpa karịa maka akara reagents na gbanwetụrụ karịa maka peptides linear kwụ ọtọ., n'ihi na ọ dịghị usoro chromatographic na-edozi ezinụlọ ọ bụla adịghị ọcha. Onye na-ahụ maka kemịkalụ na-enyocha ihe owuwu nyocha dị mgbagwoju anya ga-ejikọta HPLC na-agbanwe agbanwe na kemistri kọlụm nke abụọ., jikọta nkwenye oke na MS/MS ma ọ bụ maapụ peptide, ma tinye usoro ezubere iche dị ka nkewa chiral maka iguzosi ike n'ezi ihe stereochemical ma ọ bụ wepụrụ nha ya na mgbasa ọkụ ọtụtụ akụkụ maka mkpokọta mkpokọta.. Mgbe reagent na-enye nri ọnụ ọgụgụ ọgụgụ, Ihe akaebe a gbara agba bụ ihe na-ahapụ gị ka ị tụkwasị obi na njirimara na nkwupụta ịdị ọcha bụ ezigbo na enwere ike imepụtagharị - ọ bụghị otu pasent kacha elu..

ọba akwụkwọ nyocha na-ebi ndụ ma ọ bụ nwụọ na nkwekọ nke ogbe

A na-ewu ụlọ ọba akwụkwọ nyocha site n'ọtụtụ olulu mmiri, ọtụtụ vial, na - ma ọ bụrụ na ị na-eme atụmatụ n'ihu - ọtụtụ nza mmepụta. Batch-to-batch mgbanwe bụ ihe dị jụụ na-egbu enyo ogologo ogologo, n'ihi na ịfefe na obere adịghị ọcha ma ọ bụ akara mkpịsị aka chromatographic gbanwere na-agbanwe omume nyocha ọbụlagodi mgbe ọ bụla ogbe ọ bụla gafere ụdị ịdị ọcha ya..

Ịdọ aka ná ntị na-agbanwe agbanwe nwere akụkụ atọ:

• Ọtụtụ traceability - efere ọ bụla ma ọ bụ efere ọba akwụkwọ na-ejikọta ya na njirimara batch yana akwụkwọ nyocha nke akọwapụtara., ya mere enwere ike ị nweta nsonaazụ ọ bụla na ihe mepụtara ya.

• ntụnyere akara mkpịsị aka - A na-atụnyere nza ọhụrụ megide nza gara aga yana ụkpụrụ ntụnye aka site n'oge njide, uka, na profaịlụ adịghị ọcha, ọ bụghị naanị site na ngafe / ọdịda ịdị ọcha nọmba.

• Edekọbara data dị ọcha - ebumnobi na-egosi (chromatograms, uka ụdịdị) ewenyere ogbe ọ bụla karịa pasentị nchịkọta, ya mere, ụlọ nyocha nyocha nwere ike ikpebi ịdịgide onwe ha. Mmepụta Peptide

Iwu bara uru maka ọbá akwụkwọ buru ibu ma ọ bụ nyocha nyocha bụ isi n'aka onye mmekọ na-elele akara mkpịsị aka batch dị ka oge na-aga ma nwee ike igosipụta otu nza enyere ya si atụnyere nke kwadoro ihe mbụ gị..

Ọkwa nyocha Protein-emebi emebi: Ihe "Ezigbo zuru oke" na-adị ka mgbe ị na-esi nri

Ị nwere ike ịtụgharị ihe ndị a niile ka ọ bụrụ atụmanya dị mkpirikpi mgbe ị na-akọwapụta reagent ma ọ bụ nyochaa onye na-ebubata ya.. Na omume, ụlọ nyocha na-arụ ọrụ mmebi mmebi na-achọ ndị mmekọ ga-atụle QC n'azụ ihe enyere omenala peptide njikọ ịtụ kama inyefe otu ọnụ ọgụgụ dị ọcha.

Ihe ndị dị mkpa bụ: njirimara nke ihe karịrị uka na-emebibeghị; akara na mgbanwe nke ọ bụla kwadoro na conjugate ikpeazụ; Eji amino-acid ma ọ bụ nyocha ọkwa-map dekọ ntụkwasị obi usoro ebe ọ dị mkpa; Ụzọ nyocha orthogonal n'azụ ịdị ọcha na nkwupụta njirimara; a ule peptide ndekọ nke na-agụnye chromatograms raw na uka spectra kwa nza; na nkwụsi ike nke akara mkpịsị aka n'ofe ọba akwụkwọ ahụ.

Ikpo okwu njikọ a ma ama ga-eweta nkewa nke orthogonal njide-ọkwa, Nkwanye ọkwa MS/MS, njedebe nke endotoxin na bioburden, na otutu-larịị akwụkwọ na tebụl - n'ihi na nke ahụ bụ ihe dị iche n'etiti reagent nke na-emepụta ihuenyo dị ọcha na nke na-eji nwayọọ na-emepụta ihe ngosi ụgha n'ofe otu narị olulu mmiri..

Ntugharị igodo: Na nyocha nke mmebi protein, àgwà reagent bụ nkwenye nke nyocha. Nyochaa ụdị aha akara n'usoro nne na nna, kwado saịtị mgbanwe ọ bụla, jiri ụzọ nyocha orthogonal, ma manye ngbanwe akara mkpịsị aka cross-batch - n'ihi na agụpụtara ga-eji nwayọọ gosipụta nke ọ bụla n'ime ihe ndị a awụsara..

Nzọụkwụ ọzọ

Malite site na idetu klaasị reagent ihuenyo gị dabere n'ezie - onye nta akụkọ, nyocha, ma ọ bụ mkpụrụ - na akwụkwọ nyocha nke onye ọ bụla chọrọ. Jiri nke ahụ dị ka ntọala mgbe ị na-ebupụta, ma gwa ndị na-ebubata ngwaahịa ka ha jegharịa gị site na ụzọ orthogonal na ihe akaebe mkpisiaka ọtụtụ na-aga n'azụ ihe ị na-achọ ịgba.. Nkeji ole na ole ejiri nyochaa QC n'ihu nwere ike ịzọpụta gị na nsonaazụ nyocha enweghị ike imepụta nke na-ewe izu iji wepụ.