Christian Marie DC.

Perez February 1, 2021

MS Microbiology (Candidate)
Applicant for Research Assistant Position

This essay involves information regarding different learnings I have procured and
difficulties I have encountered during the week of training and familiarization inside the
Molecular Biology Laboratory – UST Central Lab. Gestational Diabetes Mellitus (GDM) research
project is mainly conducted in the said laboratory. Ma'am Therriz oriented me that the study
specifically sought to find a particular biomarker through protein expression analysis of seven
proteins – all of which are related to the mammalian target of rapamycin (mTOR). The proteins
are being monitored and investigated through Immunohistochemistry (IHC) (plus the ImageJ) and
Western Blotting Technique or Immunoblot. IHC is used to visualize the protein expression by
observing the normal placental tissue histology and those with confirmed GDM cases. In contrast,
Western Blot is used for validation of proteins expressed. Upon hearing these terms and other
technicalities on my first day was difficult, especially those unfamiliar to me. I then did proper
research about them to grasp the depth and the impact of the study. I learned to appreciate the
project itself and how it could improve the diagnosis of GDM among pregnant women.
So far, I was only able to observe how IHC is being done. Visually, the method looked easy.
However, the medical technologists in the lab like Ma'am Naw and Ma'am Therise told me that
it is complicated as the reagents used are sensitive. Factors like time and volume of the stains
could also affect the reaction to the tissue. Nonetheless, Ma'am Therriz has mentioned that it
only takes practice to have a better result. As per the Western Blot, Ma'am Therriz gave me two
protocols: traditional western blot and the recent one using the iBright Imaging Systems. I am
aware of the general steps of Immunoblot: (1) separation of proteins according to their molecular
weight through SDS-PAGE (2) Transfer of proteins from gel to a membrane (nitrocellulose or
PVDF) through electrophoretic transfer (3) Blocking of the membrane and then the probing of
the antibodies and (4) Detection of the antibodies. I have noticed that the traditional one seems
to be a bit tedious and requires a longer time to finish (e. g. the overnight incubation of the
primary antibody to the membrane) relative to the second protocol, which seems to provide
rapid results. I have also noticed that iBright Systems has its set reagents to be used. For example,
the reagent used in the blocking of the membrane, which I could not pinpoint what exactly it was
in the paper. Unlike in the traditional protocol, 3% skim milk, or bovine serum albumin (BSA; not
applied when sheep/goat antibodies are used) were both mentioned for the blocking method. I
have not done any blotting technique since. Henceforth, I somehow feel that doing this in
actuality will be difficult. However, I would try my definite best to learn the details and properly
conduct the said experiment.
Ma'am Therriz also gave me another two materials about protein quantification. I learned
that 96-well plates would be commonly utilized in the experiment using either Bicinchoninic Acid
(BCA) Assay or the Bradford Assay. As per my basic knowledge, I knew that both are calorimetric
assay, hence the use of protein standards to quantify the unknown protein sample
spectrophotometrically and plotting the net absorbances in a Standard Curve. Upon reading and
comparing two protocols, I have noticed that they both require several dilutions in small
quantities, requiring an advanced level in micropipetting skills.
 In four days, some of my laboratory skills were refreshed: the pipetting techniques,
computations for dilutions, and microscopy. Ma'am Therriz also reminded me of the importance
of orderliness in the lab, even in the proper placement of samples in the microcentrifuge stand
to lessen the confusion and the chance of mixing them up.
Suppose I would be given a chance to get officially accepted as a Research Assistant under
this project; I expect to be asked to carry out the abovementioned experiments according to the
project's established protocols and to master at least one of them (if not all). Moreover, I expect
to collect data and record them in the laboratory manual routinely and to input the data into
spreadsheets (when asked) to extrapolate the results into graphs. I also expect to research some
additional resources aside from what has been provided to check the facts. Overall, I expect to
expand my knowledge and laboratory experience inside a molecular biology lab.