1. What is the importance of the ammonium sulfate precipitation?

Ammonium sulfate precipitation is a method used to purify proteins by altering their

solubility; Ammonium sulfate is extremely soluble in water and so can make very
concentrated solutions, which can "salt out" proteins, causing their precipitation at
particular concentrations. This provides a convenient and simple means to
fractionate complex protein mixtures. Two distinct effects are observed: at low salt
concentrations, the solubility of the protein increases with increasing salt
concentration.

2. What is the purpose of the ninhydrin in the paper chromatography?
When chromatogram is allowed to dry, the various amino acids are invisible. The acids
can be visualized by spraying the paper with a compound called ninhydrin. Ninhydrin is
a chemical used to detect ammonia or primary and secondary amines, including amino
acids. When reacting with these free amines, a deep blue or purple color is produced.

3. Can a reverse primer start from any nucleotide? What about forward?
Primer sequences need to be chosen to uniquely select for a region of DNA, avoiding
the possibility of miss hybridization to a similar sequence nearby. A commonly used
method is BLAST search whereby all the possible regions to which a primer may bind
can be seen. The reverse primer can basically be placed to start from any nucleotide,
because it does not contain the desired sequence, which will later be expressed. The
forward primer on other hand, should be placed so that its ORF (open reading frame)
includes the whole sequence of interest, but does not cause a preterm stop codon.

4. On what principle filtration chromatography works?
Size exclusion or gel filtration chromatography (when mobile phase is aqueous) is a
method that separates molecules according to their size. Chromatography involves a
mobile phase (liquid or gas) flowing over a stationary phase (solid or liquid). For the
stationary phase generally gel beads (frequently made of dextran, agarose, or
acrylamide) are used. The liquid phase is the solvent that is found both around the
beads and in the pores of the stationary phase matrix.
Stationary and mobile phase will be mixed in column. To separate mixture of molecules,
such as proteins, the mixture is applied in a thin layer to the top of the column. After this
bottom of the column is opened and the column buffer slowly drips out. As the sample
molecules move down through the column, large molecules move faster than the small
ones this is because of structure of beads, their structure is covered with microscopic
holes of varying size. As the mobile phase carries the molecules down toward the
beats, the smallest molecules enter the in the holes and slow their movement. On the
other side intermediate molecules are too large to enter in the holes, they are trying to
enter and do not succeed and they are slowed as much. And the large molecules are
excluded from all the holes, so they travel the fastest, and on this way molecules are
separated, large molecules are firstly eluted, than intermediate and at the end the
smallest ones. But proteins cannot be completely separated by size, especially
intermediate ones.