Grid Blotting
Grid Blotting - Related Videos
08:01
Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition
0 Views •
Cited by 137 •
2011
Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these...
08:48
The Western Blot
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2023
Western Blotting is used to identify the presence of specific proteins in electrophoretically separated samples. Following separation by a technique known as sodium dodecyl sulfate polyacrylamide gel electrophoresis, or SDS-PAGE, western transfer is used to move proteins from a polyacrylamide gel onto a piece of membrane which traps the proteins in their respective locations. Next, the membranes are probed with antibodies in a process called immunboblotting. Immunoblotting uses...
01:15
Western Blotting
0 Views •
2023
Western blotting is an analytical technique for protein identification. It has various applications in immunology and medicine, including detecting diseases like bovine spongiform encephalopathy, mad cow disease, and human and feline immunodeficiency virus from biological samples. The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.
07:45
Western Blotting: Sample Preparation to Detection
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Cited by 49 •
2010
Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.
04:11
5-Methylcytosine Dot Blot to Determine the Extent of DNA Methylation
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2025
This video describes the technique of dot-blot to determine the level of DNA methylation in human chondrocytes in vitro via the blotting of DNA samples on a nylon membrane and the subsequent visualization using monoclonal antibodies. This helps to determine the chondrocytes' phenotype at various stages in culture.
FAQs
Blotting time and pressure determine how much liquid is removed from the specimen-coated support. Their adjustment changes the remaining film thickness, while sample volume and humidity also contribute to the final condition of the grid. Optimizing these variables helps produce a suitable specimen layer for subsequent vitrification and microscopic analysis.
Humidity and sample volume influence the amount of liquid available when the grid is blotted and therefore affect the thickness of the remaining film. Because ice thickness contributes to specimen quality, controlling these conditions can improve consistency between grids and help preserve a film appropriate for high-resolution structural analysis.
Filter paper may contact one or both sides of the specimen-coated grid during liquid removal. This choice is part of the blotting condition that determines the final film. Comparing these configurations during optimization can help researchers control liquid thickness and improve the consistency of grids prepared for cryogenic electron microscopy.
The preparation conditions used during Grid blotting can affect where particles are distributed across the thin film and how they orient within it. These features matter because uneven distribution or a strong preferred orientation can limit the structural information obtained from images. Optimization therefore supports more useful sampling of biomolecules and molecular complexes.
A small protein or macromolecular sample is applied to a perforated support grid. Filter paper then briefly contacts one or both sides to remove excess liquid and establish the desired film thickness. The grid is rapidly plunge-frozen so the remaining film vitrifies, preserving the prepared specimen for cryogenic electron microscopy.
The procedure requires a protein or macromolecular sample, a perforated support grid, filter paper, and a rapid plunge-freezing step. Important adjustable conditions include sample volume, blotting time, pressure, humidity, and whether one or both sides receive filter-paper contact. Together, these factors determine the quality and thickness of the resulting specimen film.
Researchers use this preparation approach when they need cryogenic electron microscopy grids suitable for examining proteins, macromolecular complexes, or other biomolecular specimens. Properly optimized grids can improve image quality and support high-resolution structural analysis. The method is especially relevant when particle distribution, preferred orientation, and ice thickness affect the interpretability of microscopic data.