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

This paper reports a versatile microfluidic chip developed for on-chip crystallization of proteins through the dialysis method and in situ X-ray diffraction experiments.

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Development of a Microfluidic Chip for Protein Crystallization by the Microbatch Method

Crystallography Reports ◽

10.1134/s1063774519020226 ◽

2019 ◽

Vol 64 (2) ◽

pp. 282-286 ◽

Cited By ~ 1

Author(s):

A. M. Popov ◽

P. V. Dorovatovskii ◽

D. A. Mamichev ◽

M. A. Marchenkova ◽

A. Yu. Nikolaeva

Keyword(s):

Microfluidic Chip ◽

Protein Crystallization

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

Journal of Applied Crystallography ◽

10.1107/s0021889812036527 ◽

2012 ◽

Vol 45 (5) ◽

pp. 1061-1065 ◽

Cited By ~ 1

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.

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A web-like network of type vi collagen in human skin is revealed by immuno-electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100103978 ◽

1988 ◽

Vol 46 ◽

pp. 382-383

Author(s):

Douglas R. Keene ◽

Robert W. Glanville ◽

Eva Engvall

Keyword(s):

Electron Microscopy ◽

Monoclonal Antibody ◽

Human Skin ◽

Basement Membranes ◽

Primary Antibody ◽

Fat Cells ◽

Secondary Antibody ◽

Type Vi Collagen ◽

Dermal Matrix ◽

Immuno Electron Microscopy

A mouse monoclonal antibody (5C6) prepared against human type VI collagen (1) has been used in this study to immunolocalize type VI collagen in human skin. The enbloc method used involves exposing whole tissue pieces to primary antibody and 5 nm gold conjugated secondary antibody before fixation, and has been described in detail elsewhere (2).Biopsies were taken from individuals ranging in age from neonate to 65 years old. By immuno-electron microscopy, type VI collagen is found to be distributed as a fine branching network closely associated with (but not attached to) banded collagen fibrils containing types I and III collagen (Fig. 1). It appears to enwrap fibers, to weave between individual fibrils within a fiber, and to span the distance separating fibers, creating a “web-like network” which entraps fibers within deep papillary and reticular dermal layers (Fig. 2). Relative to that in the dermal matrix, the concentration of type VI collagen is higher around endothelial basement membranes limiting the outer boundaries of nerves, capillaries, and fat cells (Fig. 3).

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High-voltage electron microscopy of subretinal scar formation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122836 ◽

1992 ◽

Vol 50 (1) ◽

pp. 486-487

Author(s):

G.E. Korte ◽

M. Marko ◽

G. Hageman

Keyword(s):

Electron Microscopy ◽

Monoclonal Antibody ◽

Retinal Pigment Epithelium ◽

Glial Cells ◽

High Voltage ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Sodium Iodate ◽

High Voltage Electron Microscopy ◽

Voltage Electron

Sodium iodate iv. damages the retinal pigment epithelium (RPE) in rabbits. Where RPE does not regenerate (e.g., 1,2) Muller glial cells (MC) forma subretinal scar that replaces RPE. The MC response was studied by HVEM in 3D computer reconstructions of serial thick sections, made using the STEREC0N program (3), and the HVEM at the NYS Dept. of Health in Albany, NY. Tissue was processed for HVEM or immunofluorescence localization of a monoclonal antibody recognizing MG microvilli (4).

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Identification of the pulmonary syndrome hantavirus by direct and colloidal gold immune Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100169109 ◽

1994 ◽

Vol 52 ◽

pp. 274-275

Author(s):

C. D. Humphrey ◽

C.S. Goldsmith ◽

L. Elliott ◽

S.R. Zaki

Keyword(s):

Electron Microscopy ◽

United States ◽

Monoclonal Antibody ◽

Colloidal Gold ◽

Phosphotungstic Acid ◽

Uranyl Acetate ◽

Hantavirus Pulmonary Syndrome ◽

Deer Mice ◽

Immune Electron Microscopy ◽

Negative Stain

An outbreak of unexplained acute pulmonary syndrome with high fatality was recognized in the spring of 1993 in the southwestern United States. The cause of the illness was quickly identified serologically and genetically as a hantavirus and the disease was named hantavirus pulmonary syndrome (HPS). Recently, the virus was isolated from deer mice which had been trapped near the homes of HPS patients, and cultivated in Vero E6 cells. We identified the cultivated virus by negative-stain direct and colloidal gold immune electron microscopy (EM).Virus was extracted, clarified, and concentrated from unfixed and 0.25% glutaraldehyde fixed supernatant fluids of infected Vero E6 cells by a procedure described previously. Concentrated virus suspensions tested by direct EM were applied to glow-discharge treated formvar-carbon filmed grids, blotted, and stained with 0.5% uranyl acetate (UA) or with 2% phosphotungstic acid (PTA) pH 6.5. Virus suspensions for immune colloidal gold identification were adsorbed similarly to filmed grids but incubated for 1 hr on drops of 1:50 diluted monoclonal antibody to Prospect Hill virus nucleoprotein or with 1:50 diluted sera from HPS virus infected deer mice.

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