Proteomics

Basic Instrumentation

Basic Instrumentation

Handling instruments plays an important role in laboratory workingon a daily basis. The use of most instruments is almost inevitableand hence a sound knowledge of the same is required.

Learning Objectives:

After interacting with this learning object, the learner will be able to:

• Analyse the theory and the mechanism of working for different instruments.

• Familiarize with the operating conditions of the 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

Basic Instrumentation

A typical colorimeter consists of a display, a scroll foradjusting the wavelength and absorbance and acuvette holder.

Colorimetry

Basic Instrumentation

Absorbance of a sample is given by the Beer lambertslaw and is defined as the logarithmic ratio of incidentlight to reflected light that is equal to the product ofpath length, epsilon constant called coefficient ofabsorbance and concentration of the solution. Fromthe equation, one can calculate the concentration ofsolution, given intensity of the incident light,reflected light and path length value.

Colorimetry

Basic Instrumentation

Based on the color and reaction involved in theexperiments, Wavelength has to be changed beforetaking the reading. Once the instrument is set inappropriate wavelength, allow it to calibrate for30min to attain the set wavelength. Meanwhile,prepare the samples to be analysed.

Colorimetry

Basic Instrumentation

Before taking the reading for each sample the cuvetteshould be rinsed with blank.

Colorimetry

Basic Instrumentation

Plot the graph between OD at 570nm andconcentration of the sample and extrapolate the ODvalues of samples of unknown concentration to findthe concentration.

Centrifugation

Basic Instrumentation

Transfer the sample to be separated into a centrifugetube and perform centrifugation at required speed,time and temperature.

Centrifugation

Basic Instrumentation

Centrifugation works on the basis of centrifugal force whichacts away from the center. Relative centrifugal force takesthe gravity into account during separation. This force alongwith the particle density and liquid density helps in theseparation of particles. Particle with high density willsediment faster than the low density ones which are left outin supernatant. If the particles are of varying density, thendifferent layers are formed after centrifugation. Thesedimentation rate is expressed in terms of Svedberg units.

UV-Spectrophotometric Analysis

Basic Instrumentation

UV-Visible spectrophotometer has a monochromator,light source, sample holder and detector. Light fromthe source is converted to a monochromatic light ofparticular wavelength and allowed to pass through thesample. The amount of light that emerges is detectedby a detector.

UV-Spectrophotometric Analysis

Basic Instrumentation

UV-Visible spectrophotometer works on the basis ofBeer-Lambert’s law, the law relates the absorbanceand the concentration of the solution. In the equationL signifies the path length, C concentration, E(epsilon) absorption coefficient and Io and Icorrespond to the intensity of light before enteringthe solution and the intensity coming out of thesolution. The intensity of the light coming out of thecuvette decreases when the concentration of thesubstances in the cuvette increases.

Laminar Air Flow

Basic Instrumentation

Laminar air flow chamber is used to maintain aseptic condition thatcan be used for cell culture and microbiological activities. Thelaminar air flow chamber used in laboratories uses horizontal air flowtype with the air flow facing towards the user and the velocity of airflow maintained constant. The filters used are of different typesdepending on the type of sample used.

Biosafety cabinets are of 3 classes.Class 1- has HEPA filters that removes contaminants from the exhaustair. This is only for environment protection.

Class 2- for common usage in microbiological activities, this cabinethas HEPA filters for filtering the entering air and the exhaust air andprovides personal, environmental and product protection.

Class 3- for handling most pathogenic microbes especially incontainment labs. HEPA (High efficiency Particulate Air) removes99.97% of particles of size 0.3 micrometers.

Laminar Air Flow

Basic Instrumentation

UV in the cabinet is used prior to the use of cabinet tokill existing micobes in cabinet. Air flow prevents theentry of any microbes from the environment. Flowmust be switched on before opening the hood.