{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/113981"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/113981","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Specific cellular labeling of nuclear and membrane proteins with antibody alternatives: Affimer and nanobody","abstract":"Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2023-12-01","abstract_html":"Submission published under a 24 month embargo labeled &#x27;U of I Access&#x27;, the embargo will last until 2023-12-01","abstract_has_math":false,"creators":["Ren, Pin"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biophysics & Quant Biology","degree_department":null,"school":null,"contributors":["Selvin, Paul","Chemla, Yann","Luthey-Schulten, Zaida Ann","Nelson, Erik"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2022,"date_issued":"2022-04-29T21:46:02Z","date_published":"2022-04-29T21:46:02Z","updated_at":"2026-07-22T22:24:53Z","subjects":["Biology"],"languages":["en","eng"],"rights":["Copyright 2021 Pin Ren"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/113981","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Selvin, Paul","Chemla, Yann","Luthey-Schulten, Zaida Ann","Nelson, Erik"]},{"key":"dc:creator","label":"Author","values":["Ren, Pin"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2022-04-29T21:46:02Z","2024-04-29T21:47:53Z","2021-12","2021-11-30"]},{"key":"dc:type","label":"Dc Type","values":["text","Thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biophysics & Quant Biology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Biology"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en","eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2021 Pin Ren"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/113981"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2023-12-01","The student, Pin Ren, accepted the attached license on 2021-11-29 at 11:21.","The student, Pin Ren, submitted this Dissertation for approval on 2021-11-29 at 11:39.","This Dissertation was approved for publication on 2021-11-30 at 09:01.","DSpace SAF Submission Ingestion Package generated from Vireo submission #17284 on 2022-04-06 at 17:17:00","Made available in DSpace on 2022-04-29T21:46:02Z (GMT). No. of bitstreams: 5 REN-DISSERTATION-2021.pdf: 14270927 bytes, checksum: 768d2c1e2a2e29fc8e2ccbc272f5c452 (MD5) Supplementary Movie 1.tif: 217519948 bytes, checksum: d805fa44a58bf4edd3eab5443e41efe4 (MD5) Supplementary Movie 2.tif: 232828947 bytes, checksum: 7ce2beb939a16b3c7e0a6ad98d1b117d (MD5) LICENSE.txt: 4204 bytes, checksum: 1ff6f95a2aaf5ce707f09682bfe771f9 (MD5) PROQUEST_LICENSE.txt: 4550 bytes, checksum: 426bf232f2f03bb84cd4d23e362f1889 (MD5) Previous issue date: 2021-11-30","Embargo set by: Seth Robbins for item 123345 Lift date: 2024-04-29T21:46:25Z Reason: Author requested U of Illinois access only (OA after 2yrs) in Vireo ETD system","Embargo set by: Seth Robbins for item 123345 Lift date: 2024-04-29T21:47:53Z Reason: Author requested U of Illinois access only (OA after 2yrs) in Vireo ETD system","Author requested U of Illinois access only (OA after 2yrs) in Vireo ETD system","U of I Only","Antibodies have proven to be useful tools in fluorescence imaging to identify targets in cells. In large part, this is because they have specific immunoreactivity to their targets. However, they also have serious problems: cross-reactivity with non-target proteins, poor cell permeability, batch-to-batch variability, complicated production procedures, and large size (~150 kD) that makes them impermeant to the cell membrane and require onerous efforts for live cell labeling. Hence, we explore alternative binding proteins. We chose three antibody-like proteins which are all small (< 15 kD), yet have very high affinity and specificity to their targets and, if necessary, can be easily driven through the cell and nuclear membrane. For internal labeling, we use an Affimer to detect the Y537S mutant of the Estrogen Receptor alpha, and a nanobody to detect tumor suppressor protein p53, two important proteins involved in cancer. We also use a commercially available nanobody, called Spot nanobody, to detect the AMPAR and N-methyl-D-aspartate receptor (NMDAR), two important cell membrane-bound receptor proteins in the neuron synapse. In all cases, we conjugate these antibody-like proteins with organic fluorophores and deliver them into live cells by transiently permeabilizing the cells with Streptolysin O (SLO). Single molecule sensitivity is shown for each of them and important biological results are presented."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Specific cellular labeling of nuclear and membrane proteins with antibody alternatives: Affimer and nanobody"]}]}],"canonical_facts":{"dc:contributor":["Selvin, Paul","Chemla, Yann","Luthey-Schulten, Zaida Ann","Nelson, Erik"],"dc:creator":["Ren, Pin"],"dc:date":["2022-04-29T21:46:02Z","2024-04-29T21:47:53Z","2021-12","2021-11-30"],"dc:description":["Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2023-12-01","The student, Pin Ren, accepted the attached license on 2021-11-29 at 11:21.","The student, Pin Ren, submitted this Dissertation for approval on 2021-11-29 at 11:39.","This Dissertation was approved for publication on 2021-11-30 at 09:01.","DSpace SAF Submission Ingestion Package generated from Vireo submission #17284 on 2022-04-06 at 17:17:00","Made available in DSpace on 2022-04-29T21:46:02Z (GMT). No. of bitstreams: 5 REN-DISSERTATION-2021.pdf: 14270927 bytes, checksum: 768d2c1e2a2e29fc8e2ccbc272f5c452 (MD5) Supplementary Movie 1.tif: 217519948 bytes, checksum: d805fa44a58bf4edd3eab5443e41efe4 (MD5) Supplementary Movie 2.tif: 232828947 bytes, checksum: 7ce2beb939a16b3c7e0a6ad98d1b117d (MD5) LICENSE.txt: 4204 bytes, checksum: 1ff6f95a2aaf5ce707f09682bfe771f9 (MD5) PROQUEST_LICENSE.txt: 4550 bytes, checksum: 426bf232f2f03bb84cd4d23e362f1889 (MD5) Previous issue date: 2021-11-30","Embargo set by: Seth Robbins for item 123345 Lift date: 2024-04-29T21:46:25Z Reason: Author requested U of Illinois access only (OA after 2yrs) in Vireo ETD system","Embargo set by: Seth Robbins for item 123345 Lift date: 2024-04-29T21:47:53Z Reason: Author requested U of Illinois access only (OA after 2yrs) in Vireo ETD system","Author requested U of Illinois access only (OA after 2yrs) in Vireo ETD system","U of I Only","Antibodies have proven to be useful tools in fluorescence imaging to identify targets in cells. In large part, this is because they have specific immunoreactivity to their targets. However, they also have serious problems: cross-reactivity with non-target proteins, poor cell permeability, batch-to-batch variability, complicated production procedures, and large size (~150 kD) that makes them impermeant to the cell membrane and require onerous efforts for live cell labeling. Hence, we explore alternative binding proteins. We chose three antibody-like proteins which are all small (< 15 kD), yet have very high affinity and specificity to their targets and, if necessary, can be easily driven through the cell and nuclear membrane. For internal labeling, we use an Affimer to detect the Y537S mutant of the Estrogen Receptor alpha, and a nanobody to detect tumor suppressor protein p53, two important proteins involved in cancer. We also use a commercially available nanobody, called Spot nanobody, to detect the AMPAR and N-methyl-D-aspartate receptor (NMDAR), two important cell membrane-bound receptor proteins in the neuron synapse. In all cases, we conjugate these antibody-like proteins with organic fluorophores and deliver them into live cells by transiently permeabilizing the cells with Streptolysin O (SLO). 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