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Lithium Chloride: A salt of lithium that has been used experimentally as an immunomodulator.
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Protein Crystallization

JoVE 5689

Protein crystallization, obtaining a solid lattice of biomolecules, elucidates protein structure and enables the study of protein function. Crystallization involves drying purified protein under a combination of many factors, including pH, temperature, ionic strength, and protein concentration. Once crystals are obtained, the protein structure can be elucidated by x-ray diffraction and computation of an electron density model. This video introduces protein crystallization and shows a general procedure. Protein expression and purification, crystallization, and x-ray diffraction are covered in the procedure. Applications of protein crystallization include in silico drug design, binding site determination, and membrane protein structure analysis. Protein crystallization is the process of obtaining a latticed solid form of a protein. These crystals are especially valuable to structural biologists, assisting in the study of protein function. Other techniques, such as mass spec or SDS-PAGE, can only provide information on the one-dimensional structure of proteins. Protein crystallization is complemented by the techniques of recombinant protein expression and x-ray diffraction. This video will show the principles of protein crystallization, a general laboratory procedure, and several of its applications in the


 Biochemistry

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Electro-encephalography (EEG)

JoVE 5420

EEG is a non-invasive technique that can measure brain activity. The neural activity generates electrical signals that are recorded by EEG electrodes placed on the scalp. When an individual is engaged in performing a cognitive task, brain activity changes and these changes can be recorded on the EEG graph. Therefore, it is a powerful tool for cognitive scientist aiming to better understand the neural correlates associated with different aspects of cognition, which will ultimately help them devise improved treatments for patients with cognitive deficits. Here, JoVE presents a brief overview of EEG and its applications in cognitive research. First, we discuss where and how EEG signals are generated. Then, we explain the use of EEG in studying cognition along with a detailed step-by-step protocol to perform an EEG experiment. Lastly, the video reviews some specific cognitive experiments that use EEG in combination with other techniques such as functional Magnetic Resonance imaging (fMRI) or transcranial direct current stimulation (tDCS).


 Behavioral Science

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Dialysis: Diffusion Based Separation

JoVE 5684

Dialysis is a common technique used in biochemistry for separating molecules based on diffusion. In this procedure, a semipermeable membrane allows the movement of certain molecules based on size. This method can be applied to the removal of buffer, known as desalting, or exchanging buffer molecules or ions from a protein solution.

This video covers the principles of dialysis along with a general procedure.  Several applications of dialysis are reviewed, including the removal of gradient reagents following ultracentrifugation, removing detergent after a membrane protein extraction, and the reconstitution of proteins by changing the solution environment. Biochemical samples typically have high buffer concentrations that can disrupt downstream processing and analysis. Dialysis is a common, inexpensive technique used to separate molecules based on diffusion. The method utilizes a semi-permeable membrane that allows the movement of certain components, based on size. This video will show the concepts of dialysis, a general procedure, and some of its uses in biochemistry. The most important aspect of dialysis is a semi-permeable membrane, which has pores that impose a molecular weight cut-off, allowing molecules below a certain size to pass through. For example, a 10k


 Biochemistry

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Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

1Department of Computer Science and Engineering, Texas A&M University, 2Beckman Institute for Advanced Science and Technology, University of Illinois, 3Department of Electrical and Computer Engineering, Kettering University, 43Scan, 5Department of Veterinary Integrative Biosciences, Texas A&M University

JoVE 3248


 Bioengineering

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PCR: The Polymerase Chain Reaction

JoVE 5056

The polymerase chain reaction, or PCR, is a technique used to amplify DNA through thermocycling – cyles of temperature changes at fixed time intervals. Using a thermostable DNA polymerase, PCR can create numerous copies of DNA from DNA building blocks called dinucleoside triphosphates or dNTPs. There are three steps in PCR: denaturation, annealing, and elongation. Denaturation is the first step in the cycle and causes the DNA to melt by disrupting hydrogen bonds between the bases resulting in single-stranded DNA. Annealing lowers the temperature enough to allow the binding of oligonucleotide primers to the DNA template. During the elongation step DNA polymerase will synthesize new double-stranded DNA. This video provides an introduction to the PCR procedure. The basic principles of PCR are described as well as a step-by-step procedure for setting up a generalized PCR reaction. The video shows the necessary components for a PCR reaction, includes instruction for primer design, and provides helpful hints for ensuring successful PCR reactions.


 Basic Methods in Cellular and Molecular Biology

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Bacterial Transformation: The Heat Shock Method

JoVE 5059

Transformation is the process that occurs when a cell ingests foreign DNA from its surroundings. Transformation can occur in nature in certain types of bacteria. In molecular biology, transformation is artificially reproduced in the lab via the creation of pores in bacterial cell membranes. Bacterial cells that are able to take up DNA from the environment are called competent cells. In the laboratory, bacterial cells can be made competent and DNA subsequently introduced by a procedure called the heat shock method. Heat shock transformation uses a calcium rich environment provided by calcium chloride to counteract the electrostatic repulsion between the plasmid DNA and bacterial cellular membrane. A sudden increase in temperature creates pores in the plasma membrane of the bacteria and allows for plasmid DNA to enter the bacterial cell. This video goes through a step-by-step procedure on how to create chemically competent bacteria, perform heat shock transformation, plate the transformed bacteria, and calculate transformation efficiency.


 Basic Methods in Cellular and Molecular Biology

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Bacterial Transformation: Electroporation

JoVE 5060

The term “transformation” refers cellular ingestion of foreign DNA. In nature, transformation can occur in certain types of bacteria. In molecular biology, however, transformation is artificially induced through the creation of pores in the bacterial cell walls. Bacterial cells that are able to take up DNA from the environment are called competent cells. Electrocompetent cells can be produced in the laboratory and transformation of these cells can be achieve via the application of an electrical field that creates pores in the cell wall through which DNA can pass. The video explains the equipment used in electroporation such as an electroporator and electroporation cuvette. The video also goes through a step-by-step procedure about how to create electrocompetent cells and electroporate cells of interest. Prediction of the success of a transformation of an experiment, by observing the time constant, as well as the importance of removing salt from the solutions when electroporating, are also mentioned.


 Basic Methods in Cellular and Molecular Biology

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Yeast Transformation and Cloning

JoVE 5083

S. cerevisiae are unicellular eukaryotes that are a commonly-used model organism in biological research. In the course of their work, yeast researchers rely upon the fundamental technique of transformation (the uptake of foreign DNA by the cell) to control gene expression, induce genetic deletions, express recombinant proteins, and label subcellular structures.

This video provides an overview of how and why yeast transformation is carried out in the lab. The important features of yeast plasmids will be presented, along with the procedure required to prepare yeast cells to incorporate new plasmids. The presentation also includes a step-by-step protocol for the lithium acetate method of yeast transformation. Finally, examples of the many applications of this essential technique will be provided.


 Biology I

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Cell-surface Biotinylation Assay

JoVE 5647

A cell can regulate the amount of particular proteins on its cell membrane through endocytosis, following which cell surface proteins are effectively sequestered in the cytoplasm. Once within a cell, these surface proteins can be either destroyed or “recycled” back to the membrane. The cell surface biotinylation assay provides researchers with a way to study these phenomena. The technique makes use of a derivative of the small molecule biotin, which can label surface proteins and then be chemically cleaved. However, if the surface protein is endocytosed, the biotin derivative will be protected from cleavage. Thus, by analyzing the uncleaved, endocytosed biotin label, scientists can assess the amounts of internalized surface proteins.In this video, we review the concepts behind the biotinylation assay, delving into the chemical structure of the biotin derivative and the mechanism of its cleavage. This is followed by a generalized protocol of the technique, and finally, a description of how researchers are currently using it to study the dynamics of different cell surface proteins.


 Cell Biology

13031
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