Aqui, descrevemos uma configuração de pinça magnética de alta velocidade que realiza medições nanomecânicas em biomoléculas sensíveis à força na taxa máxima de 1,2 kHz. Apresentamos sua aplicação a pinos de DNA e complexos SNARE como sistemas modelo, mas também será aplicável a outras moléculas envolvidas em eventos mecanobiológicos.
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Artigo de método
Pinças magnéticas de alta velocidade para medições nanomecânicas em elementos sensíveis à força
* These authors contributed equally
Neste artigo
Resumo
Resumo
Pinças magnéticas de molécula única (MTs) têm servido como ferramentas poderosas para interrogar com força biomoléculas, como ácidos nucléicos e proteínas, e, portanto, estão prontas para serem úteis no campo da mecanobiologia. Uma vez que o método geralmente se baseia no rastreamento baseado em imagens de esferas magnéticas, o limite de velocidade no registro e análise de imagens, bem como as flutuações térmicas das contas, há muito tempo dificultam sua aplicação na observação de pequenas e rápidas mudanças estruturais em moléculas-alvo. Este artigo descreve métodos detalhados para a construção e operação de uma configuração MT de alta resolução que pode resolver a dinâmica de nanoescala e milissegundos de biomoléculas e seus complexos. Como exemplos de aplicação, experimentos com pinos de cabelo de DNA e complexos SNARE (máquinas de fusão por membrana) são demonstrados, com foco em como seus estados transitórios e transições podem ser detectados na presença de forças em escala de piconewton. Esperamos que as MTs de alta velocidade continuem a permitir medições nanomecânicas de alta precisão em moléculas que detectam, transmitem e geram forças nas células e, assim, aprofundam nossa compreensão em nível molecular da mecanobiologia.
Introdução
As células sentem e respondem ativamente a estímulos mecânicos. Ao fazer isso, muitas biomoléculas exibem propriedades dependentes da força que permitem mudanças estruturais dinâmicas. Exemplos bem apreciados incluem canais iônicos mecanossensíveis e elementos citoesqueléticos que fornecem às células informações mecânicas importantes de seu ambiente circundante.
Além disso, moléculas que mostram uma natureza única de suporte de força também podem ser consideradas mecanossensíveis em um sentido mais amplo. Por exemplo, a formação local e o derretimento de duplexes de ácidos nucleicos, bem como estruturas de ordem superior, como G-quadruplexe....
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Protocolo
Todos os materiais e equipamentos descritos neste protocolo estão listados na Tabela de Materiais. O software LabVIEW para operar a configuração MT de alta velocidade descrita abaixo, bem como os scripts MATLAB para analisar dados de amostra, são depositados no GitHub (https://github.com/ShonLab/Magnetic-Tweezers) e disponibilizados publicamente.
1. Construção de aparelhos
NOTA: O princípio geral da construção MT de alta velocidade é semelhante aos sistemas MT convencionais padrão, exceto pelo uso de uma câmera de semicondutor de óxido metálico complementar (CMOS) de alta velocidad....
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Resultados
Calibração da força
Os resultados dos dois métodos de medição de força (variância do deslocamento lateral das contas e análise do espectro de potência) diferiram em 0-2 pN (Figura 2G). De acordo com os resultados da Figura 2F, podemos alcançar de forma confiável até 30 pN com ímãs regulares de neodímio.
Transições de dois estados de um hairpin de DNA de 8 pb
Primeiramente, investigamos a nanomec.......
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Discussão
Neste trabalho, introduzimos uma configuração de espectroscopia de força de molécula única que pode observar mudanças estruturais de biomoléculas com alta precisão espaço-temporal. A câmera CMOS de alta velocidade usada adquire 1.200 quadros s−1 com resolução de 1.280 x 1.024, permitindo o rastreamento de contas de 1,2 kHz. No entanto, a velocidade das medições é atualmente limitada pelo software de rastreamento de contas, de modo que o ROI é normalmente reduzido a áreas menores em medições de alta velocidade........
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Divulgações
Os autores declaram não haver conflitos de interesse.
Agradecimentos
Este trabalho foi apoiado pela bolsa da National Research Foundation of Korea (NRF) financiada pelo governo coreano (MSIT) (NRF-2022R1C1C1012176, NRF-2021R1A4A1031754 e NRF- 2021R1A6A1A10042944). S.-H.R. foi apoiado pela subvenção NRF (2021R1C1C2009717).
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Materiais
| Nome | Empresa | Número de catálogo | Comentários |
|---|---|---|---|
| Materiais para a síntese da construção | |||
| Agarose sistema da eletroforese do gel | Escada | do ADN de Mupid-2plus | |
| Bioneer | D-1037 | ||
| nTaq polimerase | Enzynomics | P050A | |
| Jogo da purificação do PCR | LaboPass | CMR0112 | |
| PEGylated Reticulador SMCC / SM(PEG)2 | ThermoFisher Scientific | 22102 | Para SNARE– Acoplamento de DNA |
| Primer B | Bioneer | 5'-Biotina/TCGCCACCATCATTTCCA-3'Para | construção de calibração de força de 5 kbp e alças de DNA |
| Primer B_hp | IDT | 5'-Biotina/TTTTTTTTTTGTTCTCTATTT TTTTAGAGAAC /AP site/ /AP site/ TCGCCACCATCATTTCCA-3'Para | construção de grampo |
| de cabelo Primer N | Bioneer | 5'-C6Amine/CATGTGGGTGACGCGAAA-3'Para | alças de DNA |
| Primer Z | Bioneer | 5'-Azide/TCGCCACCATCATTTCCA-3'Para | alças de DNA |
| Primer Z_5k | Bioneer | 5'-Azide/TTAGAGAGTATGGGTATATGACA TCG-3'Para | construção de calibração de força de 5 kbp |
| Primer Z_hp | Bioneer | 5'-Azide/GTGGCAGCATGACACC-3'Para | construção de grampo de cabelo |
| SYBR Safe DNA Gel Stain | ThermoFisher Scientific | S33102 | |
| λ-DNA | Bioneer | D-2510 | Template strand para sequências de PCR |
| DNA para proteínas SNARE | |||
| 6× Seu SNAP-25b (2-206; maiúsculo) em pET28a | caseiro | tggcgaatgggacgcgccctgtagcggcgca ttaagcgcggcgggtgtggtggtggttacgcgca gcgtgaccgctacacttgccagcgccctagc gcccgctcctttcgctttcttccttccctttctctt /br ctcgccacgttcgccggctttccccgtcaag ctctaaatcgggctccctttagggttccg atttagtgctttacggcacctcgaccccaaa aaacttgattagggtgatggttcacgtagtg ggccatcgccctgatagacggtttcgccc tttgacgttggagtccacgttctttaatagt ggactcttgttccaaactggaacaacactca accctatctcggtctattcttttgatttta agggattttgccgatttcggcctattggtta aaaaatgagctgatttaacaaaaatttaacg c/ gaattttaacaaaatattaacgtttacaat ttcaggtggcacttttcggggaaatgtgcgcgc ggaacccctatttgtttatttttaaatac attcaaatgtatccgctcatgaattaatt cttagaaaaactcatcgagcatcaaatgaaaa ctgcaatttattcatatcaggattatcaata ccatatttttgaaaaagccgtttctgtaatg aaggagaaaactcaccgaggcagttccatag gatggcaagatcctggtatcggtctgctgctggatt ccgactcgtccaacatcaatacaacctatta atttcccctct cgtcaaaaataaggttatcaag tgagaaatcaccatgagtgacgactgaatcc ggtgagaatggcaaaagtttatgcatttctt tccagacttgttcaacaggccagccattacg ctcgtcatcaaaatcactcgcatcaaccaaaa ccgttattcattcgtgattgcgcgcctgagcga gacgaaatacgcgatcgctgttaaaaggaca attacaaacaggaatcgaatgcaaccggcgcgc aggaacactgccagcgcatcaacaatatttt cacctgaatcaggatattcttctaatacctg , gaatgctgttttcccggggatcgcagtggtg agtaaccatgcatcatcaggagtacggataa aatgcttgatggtcggaagaggcataaattc cgtcagccagtttagtgaccatctcatct gtaacatcattggcaacgctacctttgccat gtttcagaaacaactctggcgcatcgggctt cccatacaatcgatagattgtcgcacctgat tgcccgacattatcgcgagcccatttatacc catataaatcagcatccatgttggaatttaa tcgcggcctagagcaagacgtttcccgttga ats atggctcataacaccttgtattactgt ttatgtaagcagacagttttattgttcatga ccaaaatcccttaacgtgagttttcgttcca ctgagcgtcagaccccgtagaaaa ggatcttcttgagatcctttttttctgcgcgcg taatctgctgcttgcaaacaaaaaaaccacc gctaccagcggtggtttgtgccggatcaaa gagctaccaactctttttccgaaggtaactg gcttcagcagagcgcagataccaaatactgt ccttctagtgtagccgtagttaggccaccac ttcaagaactctgtagcaccgcctacatacc tcgctctgctaatcctgttaccagtggctgc tgccagtggcgataagtcgtgtcttaccggg ttggactcaagacgatagttaccggataagg cgcagcggtcgggctgaacggggttcgtg cacacagcccagcttggagcgaacgacctac accgaactgagatacctacagcgtgagctat gagaaagcgccacgcttcccgaagggagaaa ggcggacaggtatccggtaagcggcagggtc ggaacaggagagcgcacgagggagcttcca gggggaaacggcg. cctggtatctttatagtcctgt cgggtttcgccacctctgacttgagcgtcga tttttgtgatgctcgtcaggggcggagcc tatggaaaaacgccagcaacgcggccttttttt acggttcctggccttttgctggccttttgctgct cacatgttctttcctgcgttatcccctgatt ctgtggataaccgtattaccgcctttgagtg agctgataccgctcgccgccgccgaacgacc gagcgcagcgagtcagtgagcgaggaagcgg aagagcgcctgatgcgtattttctccttac gcat. ctgtgcggtatttcacaccgcatatat ggtgcactctcagtacaatctgctgctgc cgcatagttaagccagtatacactccgctat cgtacgtgactgggtcatggctgcgccccg acacccgccaacacccgctgacgcgccctga cgggcttgtctgctcccccggcatccgcttaca gacaagctgtgaccgtctccgggagctgcat gtgtcagaggttttcaccgtcatcaccgaaa cgcgcgaggcagctgcggtaaagctcatcag cgtggtcgtgaagcgattcacagatgtctgc ct gttcatccgcgtccagctcgttgagtttc tccagaagcgttaatgtctggcttctgataa agcgggccatgttaagggcggttttttcctg tttggtcactgatgcctccgtgtaaggggga tttctgttcatgggggtaatgataccgatga aacgagagaggatgctcacgatacgggttac tgatgatgaacatgccccggttactggaacgt tgtgagggtaaacaactggcggtatggatgc ggcgggaccagagaaaaatcactcagggtc aatgccagcgcttcgttaatacagatgtaggt gttccacaggg tagccagcagcatcctgcga tgcagatccggaacataatggtgcagggcgcgc tgacttccggtttccagactttacgaaaca cggaaaccgaagaccattcatgttgttgctc aggtcgcagacgttttgcagcagtcgct tcacgttcgctcgcgtatcggtgattcattc tgctaaccagtaaggcaaccccgccagccta gccgggtcctcaacgacaggagcacgatcat gcgcacccgtggggccgccatgccggcgata atggcctgcttctcgccgaaacgtttggtgg cgggaccagtgacgaaggcttgagcgagggc gtgcaagattccgaataccgcaagcgacagg ccgatcatcgtcgcgctcccgaaagcgt cctcgccgaaaatgacccagagcgctgccgg cacctgtcctacgagttgcatgataaagaag acagtcataagtgcggcgacgatagtcatgc cccgcgcccaccggaaggagctgactgggtt gaaggctctcaagggcatcggtcgagatccc ggtgcctaatgagtgagctaacttacattaa ttgcgttgcgctcactgcccgctttccagtc gg GAAACCTGTCGTGCCAGCTGCATTAATGA atcggccaacgcgcggggagaggcggtttgc gtattgggcgccagggtggttttca ccagtgagacgggcaacagctgattgccctt caccgcctggccctgagagagttgcagcaag cggtccacgctggtttgccccagcaggcgaa aatcctgtttgatggtggttaacggcgggat ataacatgagctgtcttcggtatcgtcgtat cccactaccgagatatccgcaccaacgcgca gcccggactcggtaatggcgcattgcgcc cagcgccatctgatcgtcgcccgcgtgcgtgcggtgcgcgtgttgcgttggcaaccagcatc gcagtgggaacgatgccctcattcagcattt gcatggtttgttgaaaaccggacatggcact ccagtcgccttcccgttccgctatcggctga atttgattgcgagtgagatatttatgccagc cagccagacgcagacgcgccgagacagaa cttaatgggcccgctaacagcgcgatttgctgg tgacccaatgcgaccagatgctccacgccca gtcgcgtaccgtcttcatgggagaaaataat actgttgatgggttcagagacatca agaaataacgccggaacattagtgcaggcag cttccacagcaatggcatcctggtcatccag cggatagttaatgatcagcccactgacgcgt tgcgcgagaagattgtgcaccgccgctttac aggcttcgacgccgcttcgttctaccatcga caccaccacgctggcacccagttgatcggcgg cgagatttaatcgccgcgacaatttgcgacg gcgcgtgcagggccagactggaggtggcaac gccaatcagcaacgactgtttgcccgccagt tgttgtgccacgcggttgggaatgtaattca gctccgccatcgccgcttccacttttccccgccccgcttctctttcccg 5 cgttttcgcagaaacgtggctggcctggttc accacgcgggaaacggtctgataagacac cggcatactctgcgacatcgtataacgttac tggtttcacattcaccctgaattgactc tcttccgggcgctatcatgccatgccatgcgaa aggttttgcgccattcgatggtgtccgggat ctcgacgctctcccttatgcgactcctgcat taggaagcagcccagtaggttgaggccgccg ttgagcaccgccgccgcaaggaatggtgcat gcaaggagatggcgcccaacagtcccccggc · cacggcctgccaccatacccacgccgaaa caagcgctcatgagcccgaagtggcgagccc gatcttccccatcggtgatgtcggcgatata ggcgccagcaaccgcacctgtggcgccggtggtg atgccggccacgatgcgtccggcgtagagga tcgagatctcgatcccgcgaaattaatacga ctcactataggggaattgtgagcggataaca attcccctctagaaataattttgtttaactt taagaaggagatataccATGGGCAGC AGCCATCATCATCATCATCATCACA GCAGCGGCCTGGTGCCGCGC GGCAGC. ccatactagcggagat ATCGCCGAGGACGCAGACAT GCGCAATGAGCTGGAGGAGA TGCAGAGGAGGGCTGACCAG CTGGCTGATGAGTCCCTCTGGA AAGCACCCGTCGCATGCTGC AGCTGGTTGAAGAGAGTAA GATGCTGGCATCAGGACTTT GGTTATGTTGGATGAGCAAG GCGAACAACTGGAACGCATT GAGGAAGGGATGGACCAAAT CAATAAGGACATGAAAGAAG CAGAAAAGAATTTGACGGAC CTAGGAAAATTCGCCGGCCT TGCCGTGGCCCCCbr/ TGCCGTGGCCCCCT ccgccaac AAGCTTAAATCCAGTGATGC TTACAAAAAAGCCTGGGGC AATAATCAGGATGGAGTAGT GGCCAGCCAGCCTGCCG TGTGGTGGATGAACGGGAG CAGATGGCCATCAGTGGTG GCTTCATCCGCAGGGTAAC AAATGATGCCCGGGAAAAT GAGATGGATGAGAACCTG GAGCAGGTGAGCGGCATC ATCGGAAACCTCCGCCAC ATGGCTCTAGACATGGGCA ATGAGATTGACACCCAGA ATCGCCAGATCGACAGGA TCATGGAGAAGGCTGATT CCAACAAAACCAGAATTG ATGAAGCCAACCAACGTG CAACAAAGATGCTGGGAA GTGGTTAAggatccgaattcgag ctccgtcgacaagcttgcggccgcactc gagcaccaccaccaccaccactgagat ccggctgctaacaaagcccgaaagga agctgagttggctgctgccaccgctgag caataactagcataaccccttggggcct ctaaacgggtcttgaggggttttttgctga aaggaggaactatatccggat | |
| 6× VAMP2 marcado com His (2-97, L32C/I97C; maiúsculo) em pET28a | caseiro | tggcgaatgggacgcgccctgtagcggcgca ttaagcgcggcgggtggtggtggttacgcgca gcgtgaccgctacacttgccagcgccctagc gcccgctccttttttcttcccttcctttbr/ gcccgctccttttcttccttcttctccttt ctcgccacgttcgccggctttccccgtcaag ctctaaatcgggctccctttagggttccg atttagtgctttacggcacctcgaccccaaa aaacttgattagggtgatggttcacgtagtg ggccatcgccctgatagacggtttcgccc ggactcttgttccaaactggaacaacactca accctatctcggtctattcttttgatttta agggattttgccgatttcggcctattggtta aaaaatgagctgatttaacaaaaatttaacg c/ gaattttaacaaaatattaacgtttacaat ttcaggtggcacttttcggggaaatgtgcgcgc ggaacccctatttgtttatttttaaatac attcaaatgtatccgctcatgaattaatt cttagaaaaactcatcgagcatcaaatgaaaa ctgcaatttattcatatcaggattatcaata ccatatttttgaaaaagccgtttctgtaatg aaggagaaaactcaccgaggcagttccatag gatggcaagatcctggtatcggtctgctgctggatt ccgactcgtccaacatcaatacaacctatta atttcccctct cgtcaaaaataaggttatcaag tgagaaatcaccatgagtgacgactgaatcc ggtgagaatggcaaaagtttatgcatttctt tccagacttgttcaacaggccagccattacg ctcgtcatcaaaatcactcgcatcaaccaaaa ccgttattcattcgtgattgcgcgcctgagcga gacgaaatacgcgatcgctgttaaaaggaca attacaaacaggaatcgaatgcaaccggcgcgc aggaacactgccagcgcatcaacaatatttt cacctgaatcaggatattcttctaatacctg , gaatgctgttttcccggggatcgcagtggtg agtaaccatgcatcatcaggagtacggataa aatgcttgatggtcggaagaggcataaattc cgtcagccagtttagtgaccatctcatct gtaacatcattggcaacgctacctttgccat gtttcagaaacaactctggcgcatcgggctt cccatacaatcgatagattgtcgcacctgat tgcccgacattatcgcgagcccatttatacc catataaatcagcatccatgttggaatttaa tcgcggcctagagcaagacgtttcccgttga ats atggctcataacaccttgtattactgt ttatgtaagcagacagttttattgttcatga ccaaaatcccttaacgtgagttttcgttcca ctgagcgtcagaccccgtagaaaa ggatcttcttgagatcctttttttctgcgcgcg taatctgctgcttgcaaacaaaaaaaccacc gctaccagcggtggtttgtgccggatcaaa gagctaccaactctttttccgaaggtaactg gcttcagcagagcgcagataccaaatactgt ccttctagtgtagccgtagttaggccaccac ttcaagaactctgtagcaccgcctacatacc tcgctctgctaatcctgttaccagtggctgc tgccagtggcgataagtcgtgtcttaccggg ttggactcaagacgatagttaccggataagg cgcagcggtcgggctgaacggggttcgtg cacacagcccagcttggagcgaacgacctac accgaactgagatacctacagcgtgagctatg agaaagcgccacgcttcccgaagggagaaa ggcggacaggtatccggtaagcggcagggtc ggaacaggagagcgcacgagggagcttcca gggggaaacggcg cctggtatctttatagtcctgt cgggtttcgccacctctgacttgagcgtcga tttttgtgatgctcgtcaggggcggagcc tatggaaaaacgccagcaacgcggccttttt acggttcctggccttttgctggccttttgctgct cacatgttctttcctgcgttatcccctgatt ctgtggataaccgtattaccgcctttgagtg agctgataccgctcgccgccgccgaacgacc gagcgcagcgagtcagtgagcgaggaagc ggaagagcgcctgatgcggtattttctccttac gcatcat. ctgtgcggtatttcacaccgcatatat ggtgcactctcagtacaatctgctctctgc cgcatagttaagccagtatacactccgctat cgtacgtgactgggtcatggctgcgccccg acacccgccaacacccgctgacgcgccctga cgggcttgtctgctcccccggcatccgcttaca gacaagctgtgaccgtctccgggagctgcat gtgtcagaggttttcaccgtcatcaccgaaa cgcgcgaggcagctgcggtaaagctcatcag cgtggtcgtgaagcgattcacagatgtctgc ct gttcatccgcgtccagctcgttgagtttc tccagaagcgttaatgtctggcttctgataa agcgggccatgttaagggcggttttttcctg tttggtcactgatgcctccgtgtaaggggga tttctgttcatgggggtaatgataccgatga aacgagagaggatgctcacgatacgggttac tgatgatgaacatgccccggttactggaacgt tgtgagggtaaacaactggcggtatggatgc ggcgggaccagagaaaaatcactcagggtc aatgccagcgcttcgttaatacagatgtaggt gttccacaggg tagccagcagcatcctgcga tgcagatccggaacataatggtgcagggcgcgc tgacttccggtttccagactttacgaaaca cggaaaccgaagaccattcatgttgttgctc aggtcgcagacgttttgcagcagtcgct tcacgttcgctcgcgtatcggtgattcattc tgctaaccagtaaggcaaccccgccagccta gccgggtcctcaacgacaggagcacgatcat gcgcacccgtggggccgccatgccggcgata atggcctgcttctcgccgaaacgtttggtgg cgggaccagtgacgaaggcttgagcgagggc gtgcaagattccgaataccgcaagcgacagg ccgatcatcgtcgcgctcccgaaagcgt cctcgccgaaaatgacccagagcgctgccgg cacctgtcctacgagttgcatgataaagaag acagtcataagtgcggcgacgatagtcatgc cccgcgcccaccggaaggagctgactgggtt gaaggctctcaagggcatcggtcgagatccc ggtgcctaatgagtgagctaacttacattaa ttgcgttgcgctcactgcccgctttccagtc gg GAAACCTGTCGTGCCAGCTGCATTAATGA atcggccaacgcgcggggagaggcggtttgc gtattgggcgccagggtggttttca ccagtgagacgggcaacagctgattgccctt caccgcctggccctgagagagttgcagcaag cggtccacgctggtttgccccagcaggcgaa aatcctgtttgatggtggttaacggcgggat ataacatgagctgtcttcggtatcgtcgtat cccactaccgagatatccgcaccaacgcgca gcccggactcggtaatggcgcattgcgcc cagcgccatctgatcgtcgcccgcgtgcgtgcggtgcgcgtgttgcgttggcaaccagcatc gcagtgggaacgatgccctcattcagcattt gcatggtttgttgaaaaccggacatggcact ccagtcgccttcccgttccgctatcggctga atttgattgcgagtgagatatttatgccagc cagccagacgcagacgcgccgagacagaa cttaatgggcccgctaacagcgcgatttgctgg tgacccaatgcgaccagatgctccacgccca gtcgcgtaccgtcttcatgggagaaaataat actgttgatgggttcagagacatca agaaataacgccggaacattagtgcaggcag cttccacagcaatggcatcctggtcatccag cggatagttaatgatcagcccactgacgcgt tgcgcgagaagattgtgcaccgccgctttac aggcttcgacgccgcttcgttctaccatcga caccaccacgctggcacccagttgatcggcgg cgagatttaatcgccgcgacaatttgcgacg gcgcgtgcagggccagactggaggtggcaac gccaatcagcaacgactgtttgcccgccagt tgttgtgccacgcggttgggaatgtaattca gctccgccatcgccgcttccacttttccccgccccgcttctctttcccg 5 cgttttcgcagaaacgtggctggcctggttc accacgcgggaaacggtctgataagacac cggcatactctgcgacatcgtataacgttac tggtttcacattcaccctgaattgactc tcttccgggcgctatcatgccatgccatgcgaa aggttttgcgccattcgatggtgtccgggat ctcgacgctctcccttatgcgactcctgcat taggaagcagcccagtaggttgaggccgccg ttgagcaccgccgccgcaaggaatggtgcat gcaaggagatggcgcccaacagtcccccggc · cacggcctgccaccatacccacgccgaaa caagcgctcatgagcccgaagtggcgagccc gatcttccccatcggtgatgtcggcgatata ggcgccagcaaccgcacctgtggcgccggtggtg atgccggccacgatgcgtccggcgtagagga tcgagatctcgatcccgcgaaattaatacga ctcactataggggaattgtgagcggataaca attcccctctagaaataattttgtttaactt taagaaggagatataccATGGGCAGC AGCCATCATCATCATCATCATCAC AGCAGCGGCCTGGTGCCGC GCGGCAGCCATATC ggcagat CTCTCGGCTACCGCTGCCAC CGTCCCGCCTGCCCCCCG GCCGGCGAGGGTGGCCCCC CTGCACCTCCTCCAAATCTTA CCAGTAACAGGAGATGCCAG CAGACCCAGGCCCAGGTGG ATGAGGTGGTGGACATCATG AGGGTGAATGTGGACAAGGT CCTGGAGCGAGACCAGAAG CTATCGGAACTGGATGATCG CGCAGATGCCCTCCAGGCA GGGGCCTCCCAGTTTGAAA CAAGTGCAGCCAAGCTCAA GCGCAAATACTGGTGGAAA AACCTCAAGATGATGTGCTA Aggatccgaattcgagctccgtcg acaagcttgcggccgcactcgagcacca ccaccaccactgagatccggctgctaacaaa gcccgaaaggaagctgagttggctgctgcca ccgctgagcaataactagcataaccttgg ggcctctaaacgggtcttgaggggttttttg ctgaaaggaggaactatatccggat | |
| 6× Marcado com a marca Δ N-VAMP2 (49– 96; em maiúsculas) e Sintaxina-1A (191– 267, I202C / I266C; em maiúsculas) in pETDuet-1 | homemade | ggggaattgtgagcggataacaattcccctc tagaaataattttgtttaactttaagaagga gatataccATGGGCAGCAGCCATCA TCATCATCATCACAGCAGCGG CCTGGAAGTTCTGTTCCAGGG GCCCGGTAATGTGGACAAGGTGT CCTGGAGCGAGACCAGAAGCT ATCGGAACTGGATGATCGCGC AGATGCCCTCCAGGCAGGC CTCCCAGTTTGAAACAAGTGC AGCCAAGCTCAAGCGCAAATAC TGGTGGAAAAACCTCAAGATGAT GTAAgcggccgcataatgcttaagtcgaaca gaaagtaatcgtattgtacacggccgcataa tcgaaattaatacgactcactataggggaat tgtgagcggataacaattccccatcttagta tattagttaagtataagaaggagatatacat ATGGCCCTCAGTGAGTGAATA gatcgaga CCAGGCACAGTGAGTGCATC AAGTTGGAGAACAGCATCCG GGAGCTACACGATATGTTCAT GGACATGGCCATGCTGGTGG AGAGCCAGGGGGAGATGATT GACAGGATCGAGTACAATGTG GAACACGCTGTGGACTACGTG GAGAGGGCCGTGTCTGACACC AAGAAGGCCGTCAAGTACCAG AGCAAGGCACGCAGGAAGAA GTGCATGATCTAActcgagtc tggtaaagaaaccgctgctgcgaaatttgaa cgccagcacatggactcgtcgtc. tactagcgcag cttaattaacctaggctgctgccaccgctga gcaataactagcataaccccttggggcctct aaacgggtcttgaggggttttttgctgaaag gaggaactatatccggattggcgaatgggac gcgccctgtagcggcggcgcgg gtgtggtggttacgcgcagcgtgaccgctac acttgccagcgccctagcgcccgctcctttc gctttcttccccctttctcgccacgttcg ccggctttccccgtcaagctctaaatcgggg gctccctttagggttgtagtgcttta cggcacctcgaccccaaaaaacttgattagg gtgatggttcacgtagtgggccatcgccctg atagacggtttcgccctttgacgttggag tccacgttctttaatagtggactcttgttcc aaactggaacaacactcaaccctatctcggt ctattcttttgatttataagggattttgccg atttcggcctattggttaaaaaatgagctga tttaacaaaaatttaacgcgaattttaacaa aatattaacgtttacaatttctggcggcacg atggcatgagattatcaaaaaaggatcttcac ctagac tccttttaaattaaaaatgaagtttt aaatcaatctaagtatatgagtaaactt ggtctgacagttaccaatgcttaatcagtgagtga ggcacctatctcagcgatctgtctatttcgt tcatccatagttgcctgactccgtcgtgtgt agataactacgatacgggagggcttaccatc tggccccagtgctgcaatgataccgcgagac ccacgctcaccggctccagatttatcagcaa taaaccagccagccggaagggccgagcgca gaagtggtcctgcaactttatccgcctctccatc cagtct attaattgttgccgggaagctagag taagtagttcgccagttaatagtttgcgcaaa cgttgttgccattgctacaggcatcgtggtggtggtg tcacgctcgtcgtgtgtatggcttcattca gctccggttcccaacgatcaaggcgagttac atgatcccccatgttgtgcaaaaaagcggtt agctccttcggtcctccgatcgttgtcagaa gtaagttggccgcagtgttatcactcatggt tatggcagcactgcataattctcttactgtc atgccatccgtaagatgcttttctgtgactg gtgac gtactcaaccaagtcattctgagaata gtgtatgcggcgaccgagttgctcttgccg gcgtcaatacgggataataccgcgccacata gcagaactttaaaagtgctcattggaaa acgttcttcggggcgaaaactctcaaggatc ttaccgctgttgagatccagttcgatgtaac ccactcgtgcacccaactgatcttcagcatc ttttactttcaccagcgtttctgggtgagcaaaa aacaggaaggcaaaatgccgcaaaaagg gaataagggcgacacggaaatgttgaatact catactcttcctttttcaatcatgattgaag catttatcagggttattgtctcatgagcgga tacatatttgaatgtatttagaaaaataaac aaataggtcatgaccaaaatcccttaacgtg agttttcgttccactgagcgtcagaccccgt Agiaaagatcaaaggatcttcttgagatcct ttttttctgcgcgtaatctgctgcttgcaaa caaaaaaaccgctaccagcggtggtttg tttgccggatcaagagctaccaactcttttt ccgaaggtaactggcttcagcagagcgcagaga / taccaaatactgtccttctagtgtagccgta gttaggccaccacttcaagaactctgtagca ccgcctacatacctcgctgctgctaatcctgt taccagtggctgctgccagtggcgataagtc gtgtcttaccgggttggactcaagacgatag ttaccggataaggcgcagcggtcgggctgaa cggggggttcgtgcacacagcccagcttgga gcgaacgacctacaccgaactgagataccta cagcgtgagctatgagaaagcgccacgcttccc gaagggagaaaggcggacaggtatccggta ag cggcagggtcggaacaggagagcgcac gagggagcttccagggaaacgcctggtatc tttatagtcctgtcgggtttcgccacctctg acttgagcgtcgatttttgtgatgctcgtca ggggggcggagcctatggaaaaacgccagc aacgcggcctttttacggttcctggccttttg ctggccttttgctcacatgttctttcctgcgcg ttatcccctgattctgtgtggataaccgtatta ccgcctttgagtgagctgataccgctcgccgccgc agccgaacgaccgagcgcagcgagtcagtg agcgaggaagcggaagagcgcctgatgcgg tattttctccttacgcatctgtgcggtatttc acaccgcatatatggtgcactctcagtacaa tctgctctctgatgccgcatagttaagccta tacactccgctatcgtacgtgactgggtca tggctgcgccccgacacccgcaacacccgc tgacgcgccctgacgggcttgtctgctcccccg gcatccgcttacagacaagctgtgaccgtct ccgggagctgcatgtgtcagaggttttcacc gtcatcaccgaaacgcgcgaggcgcctgcgg · taaagctcatcagcgtggtcgtgaagcgatt cacagatgtctgcctgttcatccgcgtccag ctcgttgagtttctccagaagcgttaatgtc tggcttctgataaagcgggccatgttaaggg cggttttttcctgtttggtcactgatgcctc cgtgtaagggatttctgttcatgggggta atgataccgatgaaacgagagaggatgctca cgatacgggttactgatgatgaacatgcccg gttactggaacgttgtgagggtaaacaactg gcggtatggatgcggcgggaccagagaaaa tc actcagggtcaatgccagcgcttcgttaa tacagatgtaggtgttccacagggtagccag cagcatcctgcgatgcagatccggaacataa tggtgcagggcgctgacttccggtttccag actttacgaaacacggaaaccgaagaccatt catgttgttgctcaggtcgcagacgttttgc agcagcagtcgcttcacgttcgctcgcgtat cggtgattcattctgctaaccagtaaggcaa ccccgccagcctagccgggtcctcaacgaca ggagcacgatcatgctagtcatgccccgcgcgc , ccaccggaaggagctgactgggttgaaggct ctcaagggcatcggtcgagatcccggtgcct aatgagtgagctaacttacattaattgcgtt gcgctcactgcccgctttccagtcgggaaac ctgtcgtgccagctgcattaatgaatcggcc aacgcgcggggagaggcggtttggtattgg gcgccagggtggtttttcttttcaccagtga gacgggcaacagctgattgcctcaccgcc tggccctgagagagttgcaagcaagcggtcca cgctggtttgccccagcaggcgaaaatcctg , tttgatggtggttaacggcgggatataacat gagctgtcttcggtatcgtcgtatccca ccgagatgtccgcaccaacgcgcagcgcccgga ctcggtaatggcgcgcattgcgcccagcgcc atctgatcgttggcaaccagcatcgcagtgg gaacgatgccctcattcagcatttgcatggt ttgttgaaaaccggacatggcactccagtcg ccttcccgttccgctatcggctgaatttgat tgcgagtgagatatttatgccagccagccagccag acgcagacgcgccgagacagaacttaatggg cgcgagtaacgacgtaacgacgcgtaagcgtaacgcgcgtaacagtaagcgtagtaagcgtagtgccatgcgaatgccatgcgaacgccatgccgcgccgccatgcgccgcgcgtcgctatcgcgtcgcgtcgctatcgcgtcgcgtccgctaacagcgcgatttgctggtgaccca atgcgaccagatgctccacgcccagtcgcgt accgtcttcatgggagaaaataatactgttg atgggtgtctggtcagagacatcaagaaata acgccggaacattagtgcaggcagcttccac agcaatggcatcctggtcatccagcggatag ttaatgatcagcccactgacgcgttgcgcgcga gaagattgtgcaccgccgctttacaggcttc gacgccgcttcgttctaccatcgacaccaccacc acgctggcacccagttgatcggcgcgagatt taatcggac. ccgcgacaatttgcgacggcgcgtg cagggccagactggaggtggcaacgccaatc agcaacgactgtttgcccgccagttgttgtg ccacgcggttgggaatgtaattcagctccgc catcgccgcttccactttttcccgcgttttc gcagaaacgtggctggcctggttcacgc gggaaacggtctgataagagacaccggcata ctctgcgacatcgtataacgttactggtttc acattcaccaccctgaattgactctcttccg ggcgctatcatgccataccgcgaaaggtttt gcgg. ccattcgatggtgtccgggatctcgacg ctctcccttatgcgactcctgcattaggaag cagcccagtaggttgaggccgttgagca ccgccgccgcaaggaatggtgcatgcaagga gatggcgcccaacagtcccccggccacgggg cctgccaccatacccacgccgaaacaagcgc tcatgagcccgaagtggcgagcccgatcttc cccatcggtgatgtcggcgatataggcgcca gcaaccgcacctgtggcgccggtgatgccgg ccacgatgcgtccggcgtagaggatcgagat cgatctcgatcccgcgaaattaatacgactc actata | |
| SNAP-25b (1– 206, todos C a A; em maiúsculas) em pET28a | caseiro | tggcgaatgggacgcgccctgtagcggcgca ttaagcgcggcgggtgtggtggttacgcgca gcgtgaccgctacacttgccagcgccctagc gcccgctcctttctcgctttcttccctcttt ctcgccaccac gttcgccggctttccccgtcaag ctctaaatcgggctccctttagggttccg atttagtgctttacggcacctcgaccccaaa aaacttgattagggtgatggttcacgtagtg ggccatcgccctgatagacggtttttcgccc tttgacgttggagtccacgttctttaatagt ggactcttgttccaaactggaacaacactca accctatctcggtctattcttttgatttta agggattttgccgatttcggcctattggtta aaaaatgagctgatttaacaaaaatttaacg c/ gaattttaacaaaatattaacgtttacaat ttcaggtggcacttttcggggaaatgtgcgcgc ggaacccctatttgtttatttttaaatac attcaaatgtatccgctcatgaattaatt cttagaaaaactcatcgagcatcaaatgaaaa ctgcaatttattcatatcaggattatcaata ccatatttttgaaaaagccgtttctgtaatg aaggagaaaactcaccgaggcagttccatag gatggcaagatcctggtatcggtctgctgctggatt ccgactcgtccaacatcaatacaacctatta atttcccctct cgtcaaaaataaggttatcaag tgagaaatcaccatgagtgacgactgaatcc ggtgagaatggcaaaagtttatgcatttctt tccagacttgttcaacaggccagccattacg ctcgtcatcaaaatcactcgcatcaaccaaaa ccgttattcattcgtgattgcgcgcctgagcga gacgaaatacgcgatcgctgttaaaaggaca attacaaacaggaatcgaatgcaaccggcgcgc aggaacactgccagcgcatcaacaatatttt cacctgaatcaggatattcttctaatacctg , gaatgctgttttcccggggatcgcagtggtg agtaaccatgcatcatcaggagtacggataa aatgcttgatggtcggaagaggcataaattc cgtcagccagtttagtgaccatctcatct gtaacatcattggcaacgctacctttgccat gtttcagaaacaactctggcgcatcgggctt cccatacaatcgatagattgtcgcacctgat tgcccgacattatcgcgagcccatttatacc catataaatcagcatccatgttggaatttaa tcgcggcctagagcaagacgtttcccgttga ats atggctcataacaccttgtattactgt ttatgtaagcagacagttttattgttcatga ccaaaatcccttaacgtgagttttcgttcca ctgagcgtcagaccccgtagaaaa ggatcttcttgagatcctttttttctgcgcgcg taatctgctgcttgcaaacaaaaaaaccacc gctaccagcggtggtttgtgccggatcaaa gagctaccaactctttttccgaaggtaactg gcttcagcagagcgcagataccaaatactgt ccttctagtgtagccgtagttaggccaccac ttcaagaactctgtagcaccgcctacatacc tcgctctgctaatcctgttaccagtggctgc tgccagtggcgataagtcgtgtcttaccggg ttggactcaagacgatagttaccggataagg cgcagcggtcgggctgaacggggttcgtg CacaGCCCCAGCTTGGAGCGAACGACCTAC ACGAACTGAGATACCTACAGCGTGAGCTATG AGAAAGCGCCgCTTCCCGAAGGGAGAAA GGGGGACAGGTATCCGGTAAGCGGCAGGGTC GGAACAGGAGGCagagagCGGgagCCTcc AGGGGAAACaacBR gcctggtatctttatagtcctgt cgggtttcgccacctctgacttgagcgtcgtcga tttttgtgatgctcgtcaggggcggagcc tatggaaaaacgccagcaacgcggccttttttt acggttcctggccttttgctggccttttgctgct cacatgttctttcctgcgttatcccctgatt ctgtggataaccgtattaccgcctttgagtg agctgataccgctcgccgccgccgaacgacc gagcgcagcgagtcagtgagcgaggaagc ggaagagcgcctgatgcggtattttctccttac gcatcat. ctgtgcggtatttcacaccgcatatat ggtgcactctcagtacaatctgctctctgc cgcatagttaagccagtatacactccgctat cgtacgtgactgggtcatggctgcgccccg acacccgccaacacccgctgacgcgccctga cgggcttgtctgctcccccggcatccgcttaca gacaagctgtgaccgtctccgggagctgcat gtgtcagaggttttcaccgtcatcaccgaaa cgcgcgaggcagctgcggtaaagctcatcag cgtggtcgtgaagcgattcacagatgtctgc ct gttcatccgcgtccagctcgttgagtttc tccagaagcgttaatgtctggcttctgataa agcgggccatgttaagggcggttttttcctg tttggtcactgatgcctccgtgtaaggggga tttctgttcatgggggtaatgataccgatga aacgagagaggatgctcacgatacgggttac tgatgatgaacatgccccggttactggaacgt tgtgagggtaaacaactggcggtatggatgc ggcgggaccagagaaaaatcactcagggtc aatgccagcgcttcgttaatacagatgtaggt gttccacaggg tagccagcagcatcctgcga tgcagatccggaacataatggtgcagggcgcgc tgacttccggtttccagactttacgaaaca cggaaaccgaagaccattcatgttgttgctc aggtcgcagacgttttgcagcagtcgct tcacgttcgctcgcgtatcggtgattcattc tgctaaccagtaaggcaaccccgccagccta gccgggtcctcaacgacaggagcacgatcat gcgcacccgtggggccgccatgccggcgata atggcctgcttctcgccgaaacgtttggtgg cgggaccagtgacgaaggcttgagcgagggc gtgcaagattccgaataccgcaagcgacagg ccgatcatcgtcgcgctcccgaaagcgt cctcgccgaaaatgacccagagcgctgccgg cacctgtcctacgagttgcatgataaagaag acagtcataagtgcggcgacgatagtcatgc cccgcgcccaccggaaggagctgactgggtt gaaggctctcaagggcatcggtcgagatccc ggtgcctaatgagtgagctaacttacattaa ttgcgttgcgctcactgcccgctttccagtc gg GAAACCTGTCGTGCCAGCTGCATTAATGA atcggccaacgcgcggggagaggcggtttgc gtattgggcgccagggtggttttca ccagtgagacgggcaacagctgattgccctt caccgcctggccctgagagagttgcagcaag cggtccacgctggtttgccccagcaggcgaa aatcctgtttgatggtggttaacggcgggat ataacatgagctgtcttcggtatcgtcgtat cccactaccgagatatccgcaccaacgcgca gcccggactcggtaatggcgcattgcgcc cagcgccatctgatcgtcgcccgcgtgcgtgcggtgcgcgtgttgcgttggcaaccagcatc gcagtgggaacgatgccctcattcagcattt gcatggtttgttgaaaaccggacatggcact ccagtcgccttcccgttccgctatcggctga atttgattgcgagtgagatatttatgccagc cagccagacgcagacgcgccgagacagaa cttaatgggcccgctaacagcgcgatttgctgg tgacccaatgcgaccagatgctccacgccca gtcgcgtaccgtcttcatgggagaaaataat actgttgatgggttcagagacatca agaaataacgccggaacattagtgcaggcag cttccacagcaatggcatcctggtcatccag cggatagttaatgatcagcccactgacgcgt tgcgcgagaagattgtgcaccgccgctttac aggcttcgacgccgcttcgttctaccatcga caccaccacgctggcacccagttgatcggcgg cgagatttaatcgccgcgacaatttgcgacg gcgcgtgcagggccagactggaggtggcaac gccaatcagcaacgactgtttgcccgccagt tgttgtgccacgcggttgggaatgtaattca gctccgccatcgccgcttccacttttccccgccccgcttctctttcccg 5 cgttttcgcagaaacgtggctggcctggttc accacgcgggaaacggtctgataagacac cggcatactctgcgacatcgtataacgttac tggtttcacattcaccctgaattgactc tcttccgggcgctatcatgccatgccatgcgaa aggttttgcgccattcgatggtgtccgggat ctcgacgctctcccttatgcgactcctgcat taggaagcagcccagtaggttgaggccgccg ttgagcaccgccgccgcaaggaatggtgcat gcaaggagatggcgcccaacagtcccccggc · cacggcctgccaccatacccacgccgaaa caagcgctcatgagcccgaagtggcgagccc gatcttccccatcggtgatgtcggcgatata ggcgccagcaaccgcacctgtggcgccggtggtg atgccggccacgatgcgtccggcgtagagga tcgagatctcgatcccgcgaaattaatacga ctcactataggggaattgtgagcggataaca attcccctctagaaataattttgtttaactt taagaaggagatataccATGGCCGA GGACGCAGACATGCGCAATG AGCTGGAGGAGATGCAGAGG AGGGCTGACCAGCTCT ggctga TGAGTCCCTGGAAAGCACCC GTCGCATGCTGCAGCTGGTT GAAGAGAGTAAAGATGCTGG CATCAGGACTTTGGTTATGTT GGATGAGCAAGGCGAACAAC TGGAACGCATTGAGGAAGGG ATGGACCAAATCAATAAGGAC ATGAAAGAAGCAGAAAAGAAT TTGACGGACCTAGGAAAATTC GCCGGCCTTGCCGTGGCCCC CGCCAACAAGCTTAAATCCAG TGATGCTTACAAAAAAAGCCTG GGGCAATAATCAGGATGGAGT AGTGGCCAGCCAGCCTGCCC GTGTGGTGGATGAACGGGAG CAGATGGCCATCAGTGGTGGC TTCATCCGCAGGGTAACAAAT GATGCCCGGGAAAATGAGATG GATGAGAACCTGGAGCAGGT GAGCGGCATCGGAAACCT CCGCCACATGGCTCTAGACAT GGGCAATGAGATTGACACCCA GAATCGCCAGATCGACAGGAT CATGGAGAAGGCTGATTCCAA CAAAACCAGAATTGATGAAGC CAACCAACGTGCAACAAAGAT GCTGGGAAGTGGTTAA< > br/ ctcgagcaccaccaccaccaccactgag atccggctgctaacaaagcccgaaagga agctgagttggctgctgccaccgctgagc aataactagcataaccccttggcctc taaacgggtcttgaggggttttttgctgaaa aggaggaactatatccggat | |
| Materiais para purificação de proteínas | |||
| 2-Mercaptoethanol | SIGMA | M3148-25ML | |
| Agar | LPS Solução | AGA500 | |
| Ampicilina, Sal de sódio | PLS | AC1043-005-00 | |
| Cloranfenicol | PLS | CR1023-050-00 | |
| Células competentes ( E. coli) | Novagen | 70956 | Rosetta(DE3)pLysS |
| Glicerol | SIGMA | G5516-500ML | |
| HEPES | SIGMA | H4034-100G | |
| Ácido clorídrico / HCl | SIGMA | 320331-500ML | |
| Imidazol | SIGMA | I2399-100G | |
| Isopropil β-D-1-tiogalactopiranosídeo / IPTG | SIGMA | ||
| Sulfato de canamicina | PLS | KC1001-005-02 | |
| Luria-Bertani (LB) Caldo | LPS Solução | LB-05 | |
| Resina Ni-NTA | Qiagen | 30210 | |
| PD MiniTrap G-25 (coluna de dessalinização) | Cytiva | GE28-9180-07 | Para obter instruções, consulte: https://www.cytivalifesciences.com/en/us/shop/chromatography/prepacked-columns/desalting-and-buffer-exchange/pd-minitrap-desalting-columns-with-sephadex-g-25-resin-p-06174 |
| Fluoreto de fenilmetilsulfonilo / PMSF | ThermoFisher Scientific | 36978 | |
| Plasmídeos para proteínas SNARE | clonadas internamente | N/A | Disponível a pedido |
| Cocktail | genDEPOT | P3100 | |
| Cloreto de sódio | SIGMA | S5886-500G | |
| Fosfato de sódio dibásico / Na2HPO4 | SIGMA | S7907-100G | |
| Fosfato de sódio monobásico / NaH2PO4 | SIGMA | S3139-250G | |
| Tris(2-carboxietil)fosfina / TCEP | SIGMA | C4706-2G | |
| Base de trizma | SIGMA | T1503-250G | |
| < strong>Materiais para montagem de amostras< / strong> | |||
| Biotina-PEG-SVA | LAYSAN BIO | BIO-PEG-SVA-5K-100MG & MPEG-SVA-5K-1g | Para PEGilação |
| Dibenzociclooctino-amina / DBCO-NH2 | SIGMA | 761540-10MG | Para revestimento de contas |
| Fita dupla face | 3M | 136 | Para montagem de célula de fluxo |
| Cola epóxi | DEVCON | S-208 | Para montagem de célula de fluxo |
| Lamínula de vidro para superfície inferior | VWR | 48393-251 | Retangular, 60&vezes; 24 mm, |
| lamínula de vidro | VWR | 48393-241 | Retangular, 50&vezes; 24 mm, #1.5 |
| Conta magnética | ThermoFisher Scientific | 14301 | Dynabeads M-270 Epoxy, 2.8 μ m |
| mPEG-SVA | LAYSAN BIO | mPEG-SVA 1g | Para PEGilação |
| N,N-Dimetilformamida/DMF | SIGMA | D4551-250ML | Para revestimento de grânulos |
| N-[3-(trimetoxissilil)propil]etilenodiamina | SIGMA | 104884-100ML | Para PEGilação |
| Neutravidina | ThermoFisher Scientific | 31000 | Para amarração de amostras |
| Fosfato tampolado salino / PBS, pH 7.2 | PLS | PR2007-100-00 | |
| Seringa de plástico | Norm-ject | A5 | 5 ml, ponta luer |
| Tubo de polietileno | SCI | BB31695-PE/4 | PE-60 |
| Conta de referência | SPHEROTECH | SVP-30-5 | Partículas de poliestireno revestidas com estreptavidina; 3.0-3.4 µ m |
| Agulha de seringa | Kovax | 21G-1 1/4''21 | G |
| Bomba de seringa | KD SCIENTIFIC | 788210 | |
| Equipamento para instrumento de pinça magnética | |||
| 1 eixo motorizado microtradução estágio | PI | M-126. PD1 | Para posicionamento vertical de ímãs |
| Estágio de tradução manual de 2 eixos | ST1 | LEE400 | Para alinhamento de ímãs ao eixo óptico |
| Suporte de acrílico para ímãs | DaiKwang | Precision custum order | Desenho disponível mediante solicitação |
| Frame grabber | Silício ativo | AS-FBD-4XCXP6-2PE8 | |
| Câmera CMOS de alta velocidade | Mikrotron | EoSens 3CXP | |
| Microscópio invertido | Olympus | IX73P2F-1-2 | |
| Ímãs de neodímio | Ímã LG | ND 10x10x12t | Dimensão: 10 mm &vezes; 10 mm &vezes; 12 mm; duas lentes |
| objetivas | Olympus | UPLXAPO100XO | Imersão em óleo, NA 1.45 |
| Lente objetiva nanoposicionador | City Labs | Nano-F100S | |
| Passo de rotação motor | AUTONICS | A3K-S545W | Para ímãs rotativos |
| Diodo superluminescente | QPHOTONICS | QSDM-680-2 | 680 nm |
| Software | |||
| LabVIEW | National Instruments | v20.0f1 | |
| MATLAB | MathWorks | v2021a |
Referências
- Le, S., Liu, R., Lim, C. T., Yan, J. Uncovering mechanosensing mechanisms at the single protein level using magnetic tweezers. Methods. 94, 13-18 (2016).
- Choi, H. -K., Kim, H. G., Shon, M. J., Yoon, T. -Y. High-resolution single-molecule magnetic tweezers. Ann....
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