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oai:www.ideals.illinois.edu:2142/113981 2023-07-11 col_2142_5131 col_2142_14828 com_2142_5130 com_2142_14827 com_2142_14793 com_2142_8903

Selvin, Paul Selvin, Paul Chemla, Yann Luthey-Schulten, Zaida Ann Nelson, Erik 2022-04-29T21:46:02Z 2024-04-29T21:47:53Z 2021-12 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 application/pdf en text Specific cellular labeling of nuclear and membrane proteins with antibody alternatives: Affimer and nanobody Ren, Pin 2021-11-30 Biology 2022-04-29T21:46:02Z 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. Thesis eng http://hdl.handle.net/2142/113981 Copyright 2021 Pin Ren School of Molecular & Cell Bio Biophysics & Quant Biology University of Illinois at Urbana-Champaign Dissertation Ph.D.