Proteomics Basic Instrumentation Basic Instrumentation Handling instruments plays an important role in laboratory working on a daily basis. The use of most instruments is almost inevitable and hence a sound knowledge of the same is required. Learning Objectives: After interacting with this learning object, the learner will be able to: Familiarize with the operating conditions of the instruments. Analyse the theory and the mechanism of working for different instruments. Assess the troubleshooting steps involved in the experiments. Note: The current IDD exists in two modes- interactive and automatic. Students taking lab course should select interactive (set as default), while the automatic mode may be selected for general users.
Colorimetry A typical colorimeter consists of a display, a scroll for adjusting the wavelength and absorbance and a cuvette holder.
Colorimetry Absorbance of a sample is given by the Beer lamberts law and is defined as the logarithmic ratio of incident light to reflected light that is equal to the product of path length, epsilon constant called coefficient of absorbance and concentration of the solution. From the equation, one can calculate the concentration of solution, given intensity of the incident light, reflected light and path length value.
Colorimetry Based on the color and reaction involved in the experiments, Wavelength has to be changed before taking the reading. Once the instrument is set in appropriate wavelength, allow it to calibrate for 30min to attain the set wavelength. Meanwhile, prepare the samples to be analysed.
Colorimetry Before taking the reading for each sample the cuvette should be rinsed with blank.
Colorimetry Plot the graph between OD at 570nm and concentration of the sample and extrapolate the OD values of samples of unknown concentration to find the concentration.
Centrifugation Transfer the sample to be separated into a centrifuge tube and perform centrifugation at required speed, time and temperature.
Centrifugation Centrifugation works on the basis of centrifugal force which acts away from the center. Relative centrifugal force takes the gravity into account during separation. This force along with the particle density and liquid density helps in the separation of particles. Particle with high density will sediment faster than the low density ones which are left out in supernatant. If the particles are of varying density, then different layers are formed after centrifugation. The sedimentation rate is expressed in terms of Svedberg units.
UV-Spectrophotometric Analysis UV-Visible spectrophotometer has a monochromator, light source, sample holder and detector. Light from the source is converted to a monochromatic light of particular wavelength and allowed to pass through the sample. The amount of light that emerges is detected by a detector.
UV-Spectrophotometric Analysis UV-Visible spectrophotometer works on the basis of Beer-Lambert s law, the law relates the absorbance and the concentration of the solution. In the equation L signifies the path length, C concentration, E (epsilon) absorption coefficient and Io and I correspond to the intensity of light before entering the solution and the intensity coming out of the solution. The intensity of the light coming out of the cuvette decreases when the concentration of the substances in the cuvette increases.
Laminar Air Flow Laminar air flow chamber is used to maintain aseptic condition that can be used for cell culture and microbiological activities. The laminar air flow chamber used in laboratories uses horizontal air flow type with the air flow facing towards the user and the velocity of air flow maintained constant. The filters used are of different types depending on the type of sample used. Biosafety cabinets are of 3 classes. Class 1- has HEPA filters that removes contaminants from the exhaust air. This is only for environment protection. Class 2- for common usage in microbiological activities, this cabinet has HEPA filters for filtering the entering air and the exhaust air and provides personal, environmental and product protection. Class 3- for handling most pathogenic microbes especially in containment labs. HEPA (High efficiency Particulate Air) removes 99.97% of particles of size 0.3 micrometers.
Laminar Air Flow UV in the cabinet is used prior to the use of cabinet to kill existing micobes in cabinet. Air flow prevents the entry of any microbes from the environment. Flow must be switched on before opening the hood.