Non-robotic high-throughput setup for manual assembly of nanolitre vapour-diffusion protein crystallization screens

2012 ◽  
Vol 45 (5) ◽  
pp. 1061-1065 ◽  
Author(s):  
Rostislav Skrabana ◽  
Ondrej Cehlar ◽  
Michal Novak
Keyword(s):  
Monoclonal Antibody ◽  
High Throughput ◽  
Protein Crystallization ◽  
Design And Development ◽  
Screening Process ◽  
Manual Assembly ◽  
Synchrotron Source ◽  
Peptide Antigens ◽  
Fab Fragments ◽  
Vapour Diffusion

Nanolitre-sized drops are characteristic of high-throughput protein crystallization screening. Traditionally, reliable nanolitre drop dispensing has required the use of robotics. This work describes the design and development of a protocol for the reproducible manual assembly of nanolitre-sized protein vapour-diffusion crystallization trials in a 96/192-drop format. The protocol exploits the repetitive-pipetting mode of handheld motorized pipettes together with simple tools available in standard laboratories. The method saves precious protein material without sacrificing the effectiveness of the screening process. To verify the approach, two monoclonal antibody Fab fragments were crystallized alone and in a complex with tau peptide antigens in 0.2–0.5 µl drops. Crystals grown directly from the screen conditions in sitting drops on 96-well plates diffracted up to 1.6 Å resolution on a synchrotron source. The results proved that successful crystallization in nanolitre high-throughput format is affordable even in the absence of expensive robotic instrumentation.

Fast preparation of a desiccant array for the gradual desiccation method in protein crystallization screening

2013 ◽  
Vol 46 (3) ◽  
pp. 817-822 ◽  
Author(s):  
Qin-Qin Lu ◽  
Xu-Zhuo Xie ◽  
Yong-Ming Liu ◽  
Hui-Meng Lu ◽  
Da Chen ◽  
...  
Keyword(s):  
Aqueous Solution ◽  
High Throughput ◽  
Protein Crystallization ◽  
Protein Crystals ◽  
Diffusion Method ◽  
Screening Process ◽  
Vapor Diffusion ◽  
High Throughput Method ◽  
Droplet Array

The gradual desiccation method (GDM) is a modification of the vapor diffusion method for protein crystallization screening. This method can dramatically increase the chances of obtaining protein crystals and is therefore potentially useful for practical protein crystallization screening. However, it is troublesome to prepare the desiccant for the GDM because each of the 96 desiccants must be of the same mass. Repeated manual weighing of the desiccant (at least 96 times for one plate) to obtain the same amount is required, and manual distribution of the weighed desiccants to the respective reservoir wells is also necessary. These procedures require a considerable amount of labor and thus lower the efficiency of the screening process. Additionally, they reduce the applicability of this method in routine protein crystallization screening. To solve this problem, a high-throughput method is proposed, which involves dispensing an aqueous solution of salts (a combination of CoCl2and AlCl3) into a droplet array (8 × 12, corresponding to the arrangement in a standard crystallization plate) on a piece of tape, then drying this array to obtain the final desiccant array. Simply covering and sealing this desiccant array over the crystallization droplets in the crystallization plate can give a perfect vapor diffusion screen. With this method, the labor and automation requirements of the GDM will be comparable to those of the conventional vapor diffusion method; furthermore, the amount of the desiccant can be easily and accurately controlled, allowing the GDM to be applied in daily protein crystallization screening.

scholarly journals Easy-to-Use Osmosis-Based Microfluidic Chip for Protein Crystallization: Application to a Monoclonal Antibody

2021 ◽  
Author(s):  
Sandy Morais ◽  
Gérald Clisson ◽  
Teresa Fina Mastropietro ◽  
Maria L. Briuglia ◽  
Joop H. ter Horst ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Microfluidic Chip ◽  
Protein Crystallization

Application of a two-liquid system to sitting-drop vapour-diffusion protein crystallization

2002 ◽  
Vol 59 (1) ◽  
pp. 194-196 ◽  
Author(s):  
Hiroaki Adachi ◽  
Kazufumi Takano ◽  
Masaaki Morikawa ◽  
Shigenori Kanaya ◽  
Masashi Yoshimura ◽  
...  
Keyword(s):  
Protein Crystallization ◽  
Liquid System ◽  
Vapour Diffusion

scholarly journals A novel type of cell death of lymphocytes induced by a monoclonal antibody without participation of complement.

1995 ◽  
Vol 181 (6) ◽  
pp. 2007-2015 ◽  
Author(s):  
S Matsuoka ◽  
Y Asano ◽  
K Sano ◽  
H Kishimoto ◽  
I Yamashita ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Death ◽  
T Cell ◽  
B Cells ◽  
Interleukin 2 ◽  
Cytolytic Activity ◽  
Target Cells ◽  
T And B Cells ◽  
Fab Fragments ◽  
T Cell Clone

A monoclonal antibody, RE2, raised by immunizing a rat with cell lysate of a mouse T cell clone, was found to directly kill interleukin 2-dependent T cell clones without participation of serum complement. Fab fragments of RE2 had no cytolytic activity, while the cross-linking of Fab fragments with anti-rat immunoglobulin reconstituted the cytotoxicity. The cytotoxicity was temperature dependent: the antibody could kill target cells at 37 degrees C but not at 0 degrees C. Sodium azide, ethylenediaminetetraacetic acid, and forskolin did not affect the cytolytic activity of RE2, while the treatment of target cells with cytochalasin B and D completely blocked the activity. This suggested that the cell death involves a cytoskeleton-dependent active process. Giant holes on the cell membrane were formed within 5 minutes after the treatment with RE2, as observed by scanning electron microscopy. There was no indication of DNA fragmentation nor swelling of mitochondria during the cytolysis, suggesting that the cell death is neither apoptosis nor typical necrosis. The antibody also killed T cell lymphomas and T and B cell hybridomas only when these cells were preactivated with concanavalin A, lipopolysaccharide, or phorbol myristate acetate. Preactivated peripheral T and B cells were sensitive to the cytotoxicity of RE2, while resting T and B cells were insensitive. These results provide evidence for a novel pathway of cell death of activated lymphocytes by membrane excitation.

Construction and Evaluation of the Tumor Imaging Properties of123I-Labeled Recombinant and Enzymatically Generated Fab Fragments of the TAG-72 Monoclonal Antibody CC49

2007 ◽  
Vol 18 (3) ◽  
pp. 677-684 ◽  
Author(s):  
Ying Tang ◽  
Shaoxian Yang ◽  
Jean Gariépy ◽  
Deborah A. Scollard ◽  
Raymond M. Reilly
Keyword(s):  
Monoclonal Antibody ◽  
Tumor Imaging ◽  
Fab Fragments ◽  
Imaging Properties

scholarly journals Integration of an In Situ MALDI-Based High-Throughput Screening Process: A Case Study with Receptor Tyrosine Kinase c-MET

2017 ◽  
Vol 22 (10) ◽  
pp. 1203-1210 ◽  
Author(s):  
Katrin Beeman ◽  
Jens Baumgärtner ◽  
Manuel Laubenheimer ◽  
Karlheinz Hergesell ◽  
Martin Hoffmann ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Maldi Ms ◽  
Kinase Assay ◽  
Label Free ◽  
Screening Process ◽  
Label Free Detection ◽  
Ic50 Values

Mass spectrometry (MS) is known for its label-free detection of substrates and products from a variety of enzyme reactions. Recent hardware improvements have increased interest in the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for high-throughput drug discovery. Despite interest in this technology, several challenges remain and must be overcome before MALDI-MS can be integrated as an automated “in-line reader” for high-throughput drug discovery. Two such hurdles include in situ sample processing and deposition, as well as integration of MALDI-MS for enzymatic screening assays that usually contain high levels of MS-incompatible components. Here we adapt our c-MET kinase assay to optimize for MALDI-MS compatibility and test its feasibility for compound screening. The pros and cons of the Echo (Labcyte) as a transfer system for in situ MALDI-MS sample preparation are discussed. We demonstrate that this method generates robust data in a 1536-grid format. We use the MALDI-MS to directly measure the ratio of c-MET substrate and phosphorylated product to acquire IC50 curves and demonstrate that the pharmacology is unaffected. The resulting IC50 values correlate well between the common label-based capillary electrophoresis and the label-free MALDI-MS detection method. We predict that label-free MALDI-MS-based high-throughput screening will become increasingly important and more widely used for drug discovery.

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