Discussion:-

As a discussion protein analisis was done by SDS-Page.This method separates proteins mainly
on the basis of molecular weight as opposed to charge or folding. It is a technique that is widely
used in biochemistry, forensics, genetics and molecular biology.Isoelectric Focussing is a
method where different molecules are separated by their electric charge differences.This
technique is a type of zone electrophoresis that is usually performed in a gel and takes advantage
of the fact that a molecule’s charge changes with the pH of its surroundings.Chromatic Methods
are two methods frequently used for protein separation with high-performance liquid
chromatography and thin-layer chromatography.Both these methods are particularly useful
adjuncts to gel-based approaches.Although chromatography is a common technique in
biochemistry laboratories used for purification, identification and quantification of protein
mixtures, laser diffraction is traditionally used for pre-column size and polydispersity
management.Two-dimensional Gel Electrophoresis is a powerful gel-based method commonly
used to analyse complex samples in the interest of characterising the full range of proteins in the
sample, not just a few specific proteins.
According to the procedure,firstly we need to centrifuge at max speed of 13500rpm for 5
minutes.Centrifuging is important to separate the substances in phases according to their
molecular weight or density.The light substances will be at the top and heavy substances remains
at the bottom of the tube.Secondly,we need to decant the supernatant into waste collection
beaker.This is because to separate even smaller solid particles which may not completely settle
down with the sedimentation because all the same time during decantation,there is a chance of
the particles mixing in the liquid.
Nevertheless,we need to repeat step 1 and 2 for another round.This is because to completely mix
the substances so it will produce the cell pellet.Then,we need to completely need to remove any
residual of liquid media by pipette.This method is important to remove any residual salt during
DNA precipitation.Later on,we need to add to the cell pellet with 100 microliter of sample
buffer.Sample buffer is used to denature proteins and make them negatively charged.
Next step is,vortex the tube to mix.Vortexing is important to mix the fliud through very fast spins
and produce uniform consistency throughout the solutions.Then,we need to incubate the tube at
950C for 10 min.This is important because different bacteria like to grow at different temperature
thus microbiologist will incubate at its optimal temperature to denature the bacteria.Lastly,briefly
centrifuge the tube and keep the protein sample on ice until use.Cenrtifuging is to achieve
separation of substances in phases accoding to their molecular weight or density whereby keepin
protein on ice is to allow complete DNA resuspension.