Dicembre 7, 2023

Fluorescente: proteine ​​di fusione a catena singola Fv fluorescenti verdi

Confronto del legame cellulare e dell’assorbimento di fosfodiestere, fosforotioato e oligonucleotidi antisenso misti di fosforotioato e metilfosfonato. L’effetto delle modificazioni del fosforotioato (S-oligonucleotide) o del fosfodiestere fosforotioato terminale (SO-oligonucleotidi) o del fosfodiestere metilfosfonato (MP-O-oligonucleotidi) sul legame, assorbimento e degradazione delle cellule della milza murina è stato studiato utilizzando    oligonucleotidi coniugati con fluoresceina ( FITC  )  . Gli oligonucleotidi S hanno mostrato il legame e l’assorbimento cellulare più elevati seguiti dagli oligonucleotidi SO-, O- e MP-O-.

  1. Studi sulla concorrenza hanno dimostrato che gli S-oligonucleotidi hanno una maggiore   affinità   per i siti di legame degli oligonucleotidi di membrana poiché possono bloccare completamente il legame dell’O-oligonucleotidi con un rapporto molare di appena 0,1. L’assorbimento di tutti gli oligonucleotidi era maggiore nei linfociti B rispetto ai linfociti T ed era aumentato dalla stimolazione con il mitogeno dei linfociti B, il lipopolisaccaride.
  2. Sebbene le nostre cellule siano state   purificate   utilizzando tecniche convenzionali per eliminare le cellule morte, circa il 5% delle cellule è rimasto morto o morente come determinato dalla citometria a flusso utilizzando la colorazione con ioduro di propidio. Vale la pena notare che l’associazione degli oligonucleotidi con le cellule morte era circa 50 volte maggiore rispetto alle cellule viventi.
  3. La microscopia confocale ha confermato che gli oligonucleotidi nelle cellule vitali erano intracellulari e mostravano uno scarso assorbimento nel nucleo per 4 ore. Sebbene un’estesa degradazione degli O-oligonucleotidi intracellulari fosse visibile dopo 4 ore, non vi era alcuna degradazione rilevabile di S-, SO o MP-O-oligonucleotidi.

I cumuli di cellule mesangiali. Focolai nodulari di eccessiva crescita cellulare e matrice in coltura prolungata.

  • Per studiare la capacità delle cellule mesangiali glomerulari (MC) di produrre matrice extracellulare, gli autori hanno studiato la MC in coltura utilizzando la microscopia ottica ed elettronica, nonché l’immunocitochimica. Le MC sono state ottenute da glomeruli di ratto isolati e mantenute per un massimo di 12 settimane in un mezzo contenente il 20% di siero fetale di vitello.
  • La crescita eccessiva di MC dalla coltura primaria e fino a tre sottocolture ha mostrato un’organizzazione caratteristica costituita da filamenti di cellule allungate o stellate intrecciate. Dopo la confluenza dopo 10-16 giorni, le MC hanno continuato a crescere in multistrati irregolari. Gli MC hanno prodotto materiale di matrice extracellulare entro 2-4 giorni dopo la placcatura e grandi quantità di matrice si sono accumulate nel tempo. Dopo 2-3 settimane, focolai di eccessiva proliferazione di MC, secrezione di matrice e detriti cellulari necrotici formavano sporgenze nodulari che gradualmente formavano grandi collinette.
  • Gli studi immunocitochimici di polloni MC sono stati eseguiti su piastre di coltura o su materiale asportato utilizzando specifici anticorpi policlonali di coniglio contro proteine ​​della matrice isolate e    secondi     anticorpi coniugati con FITC  e  purificati  per  affinità .
  • Durante i 3 giorni di coltivazione, le MC hanno sviluppato fibronectina e collagene di tipo I, III, IV e V. Nel tempo, i filamenti della matrice, soprattutto nella massa centrale delle colline, si sono colorati intensamente per questi componenti. La colorazione della laminina era meno pronunciata.
  • La miosina delle cellule muscolari lisce è stata regolarmente trovata su fibre intracellulari discrete e nel materiale extracellulare delle collinette. La microscopia elettronica ha mostrato che le collinette erano costituite da cellule allungate sulla superficie e cellule stellate intervallate dalla matrice e dai detriti delle cellule necrotiche nel nucleo.
  • I risultati mostrano che le MC proliferanti possono essere mantenute in colture omogenee per periodi più lunghi. Le MC producono grandi quantità di proteine ​​della matrice extracellulare (collagene di tipo IV e V, fibronectina, laminina), che si trovano nei glomeruli normali.
  • Le MC coltivate producono anche collagene interstiziale di tipo I e III. Le colline MC mostrano un accumulo di matrice nodulare simile a quello visto nel mesangio dei glomeruli malati. Si è concluso che un modello in vitro di crescita prolungata di MC potrebbe facilitare lo studio dei fattori che regolano la produzione della matrice mesangiale. Tale modello potrebbe essere utilizzato per studiare la risposta mesangiale a specifici stimoli infiammatori o metabolici.

PET / CT in vivo per i recettori EphB4 di piccoli animali utilizzando il peptide marcato con 64Cu.

  1. Molti tumori solidi sovraesprimono il recettore EphB4, un membro della famiglia del recettore dell’efrina tirosin-chinasi. L’imaging EphB4 non invasivo ha il potenziale per aumentare i tassi di diagnosi precoce, monitorare la risposta alla terapia anti-EphB4 e migliorare i risultati dei pazienti. Lo scopo di questo studio era di valutare un nuovo (64) peptide marcato con Cu con un’elevata   affinità  per il legame dei recettori PET ai recettori EphB4.
  2. TNYLFSPNGPIARAW Il peptide legante EphB4 (TNYL-RAW) è stato coniugato con isotiocianato di fluoresceina (FITC) e DOTA. DOTA-TNYL-RAW è stato etichettato con (64) Cu con un’elevata efficienza di etichettatura. L’ affinità di legame  di TNYL-RAW e dei suoi derivati ​​per EphB4 ricombinante purificato è stata determinata utilizzando la tecnologia di risonanza plasmonica di superficie. Il legame in vitro di FITC-TNYL-RAW e (64) Cu-DOTA-TNYL-RAW alle cellule tumorali è stato valutato mediante microscopia a fluorescenza e metodi di conteggio della radioattività. La biodistribuzione in vivo e la PET/TC in piccoli animali sono state eseguite in topi portatori di tumori CT26 e PC-3M che esprimono EphB4, nonché di tumori A549 negativi per EphB4.
  3. TNYL-RAW e suoi derivati ​​hanno mostrato un’elevata   affinità di legame a EphB4, con una costante di equilibrio di dissociazione di 1,98-23 nM. In vitro, sia FITC-TNYL-RAW che (64) Cu-DOTA-TNYL-RAW sono stati assorbiti selettivamente dalle cellule CT26 e PC-3M, ma non dalle cellule A549. Il legame di FITC-TNYL-RAW e (64) Cu-DOTA-TNYL-RAW alle cellule CT26 e PC-3M può essere bloccato dall’eccesso di TNYL-RAW. In vivo, (64) Cu-DOTA-TNYL-RAW ha mostrato un assorbimento significativamente maggiore nei tumori PC-3M rispetto ai tumori A549, con una dose percentuale iniettata per grammo di tumore pari a 0,84 ± 0,09 e 0,44 ± 0,09 24 ore dopo l’iniezione del radiotracciante , rispettivamente. La PET / CT in piccoli animali ha rivelato chiaramente (64) la deposizione di Cu-DOTA-TNYL-RAW nei tumori CT26 e PC-3M, ma non nei tumori A549. Inoltre, l’assorbimento (64) di Cu-DOTA-TNYL-RAW nei tumori CT26 e PC-3M può essere bloccato dal TNYL-RAW freddo.
  • È stato progettato un sistema di espressione (pSKGFP) che consente l’espressione di frammenti variabili a catena singola come proteine ​​di fusione con proteine ​​fluorescenti verdi modificate. Questo sistema di espressione è paragonabile ai vettori di visualizzazione dei fagi comunemente usati e consente una caratterizzazione in un unico passaggio di anticorpi ricombinanti selezionati mediante citometria a flusso o colorazione cellulare fluorescente.
  • Due diversi anticorpi a frammento variabile a catena singola, entrambi diretti contro Ralstonia solanacearum lipopolisaccaridi, sono stati geneticamente fusi con una proteina fluorescente verde spostata in rosso e la proteina di fusione risultante è stata testata per l’utilità.
  • Questi fluocorpi possono essere prodotti in colture cellulari batteriche e  purificati mediante  cromatografia  per affinità con   metalli immobilizzati . Funzionano bene nella citometria a flusso e nella colorazione immunofluorescente delle cellule, sono specifici per i loro antigeni bersaglio e, a differenza  degli anticorpi coniugati  con FITC  , non sbiadiscono quando irradiati.

Caratterizzazione dell’anticorpo monoclonale anti-H-2 e suo utilizzo nella purificazione dell’antigene su larga scala.

  1. È stato riscontrato che un anticorpo monoclonale (MAb) di ratto anti-topo, M1/42, reagisce con gli antigeni H-2 degli aplotipi a, b, d, j, k, siu (tutti gli aplotipi testati). Questo anticorpo, dopo essersi legato alle cellule e aver reagito con   FITC  –    Ig anti-ratto coniugato , può essere utilizzato per quantificare l’espressione di H-2 su diversi tipi cellulari.
  2. L’anticorpo è stato anche utile per confrontare i prodotti H-2 precipitati da diversi aplotipi. Perline di sefarosio-4B accoppiate a M1/42 sono state utilizzate per purificare gli antigeni H-2d mediante cromatografia  di affinità .
  3. Molecole di H-2 pure sono state eluite dalla colonna in DOC allo 0,5%, NaCl 0,65 M, Tris 20 mM, pH 8,0, producendo da 110 a 180 microgrammi di cellule tumorali H-2d / 10 (10) P815. Questo anticorpo, utilizzato in serie con MAb specifici per H-2Kk 11-4.1, ha consentito la purificazione di Dk e Dd rispettivamente dalle cellule RDM-4 e YAC.

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10119-GAF-BLK Alpha Diagnostics 1g; bulk Ask for price

Goat Anti-Human IgA, Affinity Purified (Bulk)

10019-GAF-BLK Alpha Diagnostics 1g; bulk Ask for price

Goat Anti-Mouse IgG, Affinity Purified (Bulk)

41319-CAF-BLK Alpha Diagnostics 1g; bulk Ask for price

*Porcine Vitronectin Multimeric Affinity Purified

IPCVTNMULTIAP100UG Innovative research each 320 EUR

*Porcine Vitronectin Multimeric Affinity Purified

IPCVTNMULTIAP1MG Innovative research each 1919 EUR

Anti-Collagen III affinity purified antibody

STJ16101168 St John's Laboratory 1 mL 457.2 EUR

*Mouse IgG Affinity Purified Endotoxin Depleted

IMSIGGAPLX50MG Innovative research each 503 EUR

Goat Anti-Rabbit IgG, Affinity Purified (Bulk)

20318-GAF-BLK Alpha Diagnostics 1g; bulk Ask for price

Goat Anti-Mouse IgG1, Affinity Purified (Bulk)

40126-GAF-BLK Alpha Diagnostics 1g; bulk Ask for price

Sheep Anti-Mouse IgG, Affinity Purified (Bulk)

43319-SAF-BLK Alpha Diagnostics 1g; bulk Ask for price

Donkey Anti-Goat IgG, Affinity Purified (Bulk)

60319-DAF-BLK Alpha Diagnostics 1g; bulk Ask for price

Rabbit Anti-Goat IgG, Affinity Purified (Bulk)

60319-RAF-BLK Alpha Diagnostics 1g; bulk Ask for price

Rabbit Anti-Human IgG, Affinity Purified (Bulk)

10119-RAF-BLK Alpha Diagnostics 1g; bulk Ask for price

Sheep Anti-Rabbit IgG, Affinity Purified (Bulk)

20319-SAF-BLK Alpha Diagnostics 1g; bulk Ask for price

Donkey Anti-Sheep IgG, Affinity Purified (Bulk)

30326-CAF-BLK Alpha Diagnostics 1g; bulk Ask for price

Donkey Anti-Sheep IgG, Affinity Purified (Bulk)

30518-DAF-BLK Alpha Diagnostics 1g; bulk Ask for price

Rabbit Anti-Mouse IgG, Affinity Purified (Bulk)

44319-RAF-BLK Alpha Diagnostics 1g; bulk Ask for price

Donkey Anti-Mouse IgG, Affinity Purified (Bulk)

42319-DAF-BLK Alpha Diagnostics 1g; bulk Ask for price

Goat Anti-Mouse IgG2b, Affinity Purified (Bulk)

40123-GAF-BLK Alpha Diagnostics 1g; bulk Ask for price

Goat Anti-Mouse IgG2a, Affinity Purified (Bulk)

40127-GAF-BLK Alpha Diagnostics 1g; bulk Ask for price

Goat Anti Rat IgA Polyclonal Affinity Purified

IGTARTIGAAP1MG Innovative research each 195 EUR

Goat Anti Rat IgE Polyclonal Affinity Purified

IGTARTIGEAP500UG Innovative research each 755 EUR

Goat Anti Rat IgM Polyclonal Affinity Purified

IGTARTIGMAP1MG Innovative research each 264 EUR

Biotin Modified Affinity Purified Avidin protein

PROTP02701 BosterBio Regular: 10mg 380.4 EUR

Donkey Anti-Rabbit IgG, Affinity Purified (Bulk)

20320-DAF-BLK Alpha Diagnostics 1g; bulk Ask for price

Rabbit Anti T7 Tag Polyclonal Affinity Purified

IRBAT7TAP100UG Innovative research each 404 EUR

Rabbit Anti T7 Tag Polyclonal Affinity Purified

IRBAT7TAP1MG Innovative research each 1550 EUR

*Human IgD from Myeloma Plasma Affinity Purified

IHUIGDMYAP100UG Innovative research each 439 EUR

*Human IgD from Myeloma Plasma Affinity Purified

IHUIGDMYAP500UG Innovative research each 1144 EUR

Human IgE from Myeloma Plasma Affinity Purified

IHUIGEMYAP100UG Innovative research each 495 EUR

*Human IgM from Myeloma Plasma Affinity Purified

IHUIGMMYAP10MG Innovative research each 740 EUR

*Human IgM from Myeloma Plasma Affinity Purified

IHUIGMMYAP5MG Innovative research each 439 EUR

Multi-Species ACE2 Polyclonal Affinity Purified

IMSAMLACE2AP100ul Innovative research each 395 EUR

*Human Kallikrein Affinity Purified Biotin Labeled

IHUKLKAPBL100UG Innovative research each 461 EUR

*Human Kallikrein Affinity Purified Biotin Labeled

IHUKLKAPBL1MG Innovative research each 1627 EUR

Anti-SF9 Insect Cell HCP 2G affinity purified

SF9RB01-AF Cygnus Technologies 0.5 mg 2295.6 EUR

Rabbit anti-Mouse Moesin IgG, affinity purified

MSN11-A Alpha Diagnostics 100 ul 578.4 EUR

Rabbit Anti S-Tag Polyclonal Affinity Purified

IRBASTAGAP100UG Innovative research each 404 EUR

Rabbit Anti S-Tag Polyclonal Affinity Purified

IRBASTAGAP1MG Innovative research each 1550 EUR

Rabbit Anti AU1 Tag Polyclonal Affinity Purified

IRBAAU1TAP100UG Innovative research each 404 EUR

Rabbit Anti AU1 Tag Polyclonal Affinity Purified

IRBAAU1TAP1MG Innovative research each 1550 EUR

Rabbit Anti GST Tag Polyclonal Affinity Purified

IRBAGSTTAP100UG Innovative research each 184 EUR

Rabbit Anti GST Tag Polyclonal Affinity Purified

IRBAGSTTAP1MG Innovative research each 919 EUR

Rabbit Anti HSV Tag Polyclonal Affinity Purified

IRBAHSVTAP100UG Innovative research each 404 EUR

Rabbit Anti HSV Tag Polyclonal Affinity Purified

IRBAHSVTAP1MG Innovative research each 1550 EUR

*Human IgA1 from Myeloma Plasma Affinity Purified

IHUIGA1MYAP10MG Innovative research each 664 EUR

*Human IgA1 from Myeloma Plasma Affinity Purified

IHUIGA1MYAP5MG Innovative research each 461 EUR

*Human IgA2 from Myeloma Plasma Affinity Purified

IHUIGA2MYAP5MG Innovative research each 532 EUR

*Human IgG1 from Myeloma Plasma Affinity Purified

IHUIGG1MYAP10MG Innovative research each 666 EUR

*Human IgG1 from Myeloma Plasma Affinity Purified

IHUIGG1MYAP1MG Innovative research each 136 EUR

*Human IgG1 from Myeloma Plasma Affinity Purified

IHUIGG1MYAP5MG Innovative research each 600 EUR

Human IgG2 from Myeloma Plasma Affinity Purified

IHUIGG2MYAP5MG Innovative research each 600 EUR

*Human IgM Mu Chain Affinity Purified Lyophilized

IHUIGMMUAPLY10MG Innovative research each 1058 EUR

*Human IgM Mu Chain Affinity Purified Lyophilized

IHUIGMMUAPLY5MG Innovative research each 649 EUR

H-2d   purificato   mediante cromatografia su colonna su M1 / ​​42 è risultato sierologicamente e biologicamente attivo come determinato dal rebinding di MAb, dall’inibizione della lisi cellulare da parte di alloantisieri più complemento e dalla capacità di stimolare il CTL alloreattivo. Questo anticorpo e i protocolli descritti dovrebbero essere utili per la preparazione di quantità relativamente grandi di antigeni H-2 multipli.

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