Day 10 - Final - Thoughts

Hello, Benedict here.

I am glad to have been given this opportunity to shadow PhD students at Nanyang Technological University's  Materials and Science Engineering. It has been a very fruitful experience. It opened my eyes to both what research is about and tertiary education

In tertiary education, it it much more different from Junior College. There are no teachers there to push you to do your own work. You have to plan everything yourself, though you can still get the help of your fellow friends and the kind guidance of professors when needed. Everything from test to training means that you need to know your own subject well. Also time management is important as there will always be things for you everyday. It might be paper work like reports or hands on like doing characterisation of proteins. Overall I think it is a very independent thing.

This experience also showed me much more about research. Now when I look at things, it is from a deeper perspective, I used to wonder where these things come from.  Now I know that they are developed in labs. It is a very long process and can take years to be made. This is usually not appreciated by a layman, but now it has enlightened me. This period also showed me why research can be a long and tedious process. Although there are people who got lucky and got desired results through luck, more often than not it is through trial and error. Much similar to Thomas Edison's many trials to get the right material for the light bulb filament. Research is very methodical. If you fail, do not only try again. You need to attempt to identify what might be causing the anomly and then repeat the whole procedure. It might take long to finally narrow it down. But it will certainly be rewarding when you can finally find what you were looking for and maybe improve the life of mankind.

This programme has definitely sparked my interest in materials engineering and will always be on my mind for consideration as my future course.

Day 10 - Final Procedure with LiHong

Today is our last day and we follow both LiHong and DaWei.

First we follow LiHong.

The training on the lesson that LiHong had attened is finally put to use today.

The samples that LiHong has dissolved to be used today. They were dissolved with ethanol

Test successful for the first sample!

One of the holders for the sample.

Day 10 - Final Lab Procedure with DaWei

Next we followed DaWei

DaWei's PAGE gel which has been shown in the previous post.

It has been destained and he washed it with water.

The cleaned sample is then placed between two sheets of clear plastic

 The gel is then taken to be scanned so that the results can be saved and further analysed at his own time.

After that he takes it back to the lab. He then transfers it to a sealed plastic bag and placed in a fridge. Thus is so that he can check again on the sample should he have any doubts

Day 9

Today we follow Li Hong and her colleague Shu Hui

She introduced to us some very important Machinary which they use.

This is a HPLC (High-Performance Liquid Chromatography). This machine is very delicate in the sense that it coloumns and tubing are very fiine. Before she could put the solution that she wanted to test, she first had to purify by passing it through a syringe filter. This removes particles of certain micron. If not, it may clog up the machine which can be very costly to replace.

 This is a electronic pH metre. It is very sensitive and can measure up to 2 decimal places

Li Hong then introduced us to the NMR (Nuclear Magnetic Resonance)

Quote from Wikipedia:

''Most frequently, NMR spectroscopy is used by chemists and biochemists to investigate the properties of organic molecules, though it is applicable to any kind of sample that contains nuclei possessing spin. Suitable samples range from small compounds analyzed with 1-dimensional proton or carbon-13 NMR spectroscopy to large proteins or nucleic acids using 3 or 4-dimensional techniques. The impact of NMR spectroscopy on the sciences has been substantial because of the range of information and the diversity of samples, including solutions and solids.''

Day 8 - More on chromatography

Today we follow DaWei's friend Fu Jing on her tests. She carefully pipettes minute amounts into a small vial. Then using a centrifuge, she seperates the suspended denatured protein. The appear at the bottom of this machine.

However the interesting part of the machine is that it makes use of the centrifugal force in high speed spinning, this moves the suspended solution to the bottom of the vial. 

Next, Fu Jing uses ( 1-directional ) gel electrophoresis on the the solute which she took and dyes them. This is PAGE (Polyacrylamide Gel Electrophoresis). It eliminates then different charges of the different proteins and actually seperates them through different molecular weight. Smaller ones moves faster, while the bigger one moves faster. Thus you will be able to see different bands on the ladder.

She has to carefully pipette the solute as the ladder is very small, less than 1 cm in width and less than 0.5 mm in thickness

The gel will later be taken out in 1-2 hours time. Then it will be taken out and destained overnight in a box.

The machine controlling the voltage.

Her previous work which had already gone through PAGE.

Current work in progress.....

 Next we followed DaWei.

He took 1 micro litre of enzymes and 20 micro litre of proteins and mixes them together. The enzyme-substrate then converts the long protein chains into two separate proteins, meaning a cleavage was done here. 

Mixtures are then left overnight in a 4 degree Celsius Refrigerator. This is because the proteins are very delicate and could possibly decompose at room temperature if left overnight.

Day 7 - Following Mentor's Fellow Colleagues

We have been following our mentors for 7 days!

Today, LiHong was a little busy with her work and so, we shadowed her friend Fujing for the day. Fujing is a new PhD student and she has been working with proteins too. She brought us into the lab and showed how she was going to (hopefully) bind alpha and gamma proteins together. After she mixed both type of proteins together, she continued to carry out dialysis like what Dawei did. 

Interestingly, Paul (another researcher) came in and talked to us. He introduced two-dimensional gel electrophoresis which, is a step to separate proteins according to their isoelectric point and molecular mass. In the medical field, he said, 2D gel electrophoresis can be applied to identify unusual proteins in a brain like the analysis of brain proteins in brain cancer compared to normal brain cells. Soon after we were left wandering in the lab, observing others. 

To end off:

Research is a repetitive process and it demands patience. It could take up days to weeks or even months to obtain a desirable result. Nevertheless, the rewards are fruitful as the gain in more knowledge is truly enlightening. 

Day 6 - Some familiar steps

Today we follow LiHong on her day.

Her part of the steps will seem familiar. She does dialysis, filtration and rotary evaporation. Lastly she uses DLS (Dynamic Light Scattering) to characterise the protein.

Firstly, she completes her dialysis:

 Carefully she, removes the remaining samples still in the dialysis bag.

She then filtrates the collected solution to obtain the filtrate residue

The clear filtrate still contains some protein, so she pours it into a round-bottom flask and put in an evaporator

The residue in the filter paper will be left in the oven to dry and the solution in the rotary evaporator will be left to run until residue appears. 

 We then followed LiHong to her training. Students have to apply for them to get a licence so that they can use the machines for protein characterisation.

The Malvern Nanosizer machine with the monitor. It uses DLS

The DLS machine

During the course, she learns to start up the hardware and software, if not the computer may crash if the wrong steps are taken.

She learns how to select the appropriate apparatus to use for different conditions.

She also learns how to read graphs and recognise anomalies in the graphs. A one common problem was due to dust and the trainer taught her how to spot them. The ability to recognise graph also will tell her if she is using the appropriate equipment or she should use a different machine to characterise the protein.

Day 5 - In Pictures

Today we complete the dialysis! Then move on to freeze-drying!

The Lab: 

 Step 1: DaWei pours out the 5% acetic acid and removed urea 

Step 2: He then replaces it with a fresh solution of 5% acetic acid free of urea! 

Step3: Steps 1-2 are repeated once more at 2-3 hours each time.

Step 4: Placing it in rack and puncturing a hole. This is to ensure that the frozen water and acetic acid can be sublimed when in the freeze dryer, leaving only the protein

It it then placed in a free dryer for 1 h and 30 mins to ensure that everything is frozen.

The -80 Freezer!

Step 5: DaWei quickly places the rack containing the vial in a special glass which can withstand pressures close to vacuum. This has to be done quickly to ensure that the solution does not melt! 

   Done!

This is left overnight to ensure that all water and the 5% acetic is removed, leaving only the protein.

The freeze-dryer machine!

Our attempts successful!:

Day 5 - Completing Dialysis, onto obtaining pure denatured protein

We are halfway through our shadowing programme. Today's procedure is similar to yesterday's.

The dialysis bag containing urea, denatured protein and 5% acetic acid was further processed so that all the urea can removed from the bag. This step was repeated twice, each time roughly 2-3 hours. The result was a bag containing water, 5% acetic acid and the protein, meaning that urea has been successfully removed.

In order to obtain the pure protein powder, the bag was placed in a freezer at -80 degrees Celsius. The bag then turned solid hard. Afterwards, Dawei placed the bag in a freeze dryer. Ice and acetic acid which were initially present soon sublimed. Liquid water and aqueous acetic acid were not  formed as both substances were sublimed. 

The final product contains the protein only. 

Day 4 - Protein Extraction

Pre - Preparation: Protein (Polymer)

DaWei needs to obtain the Unfolded Protein Strand.. First he adds urea to the protein. This urea denaturises the protein into a polymer. After doing so he needs to remove the denatured protein. He then makes adds acetic acid at 5%.

Dialysis:

Step 1: Taking out the right vial containing his prepared solution.

Step 2: The bag to contain the solution is soaked in deionised water!

Step 3: DaWei proceeds to homogenise the 5% acetic acid in deionised (total 3 Litres!) water using a Homogeniser. The white motion blur you see is a magnetic mixer which is spun by a electromagnet below the machine.

Step 4: DaWei Proceeds to carefully add the protein into the bag.

Step 5: Securing the bag with clips!

Step 6: The bag is placed into a container containing roughly 800 mililitres of 5% acetic acid. This first round will be put overnight. Subsequent ones are done for 2-3 hours to remove any excess urea. Stay tuned!

How this last step works: 

The 5% acetic acid is maintained to make sure to make sure the protein stays soluble in the solution. The urea molecules are small enough to pass through the semi-permeable membrane of the bag. This is done repeatedly, removing any remaining urea.

Points to note: Dialysis although share similar steps to osmosis, they are different, to understand do check out a website by clicking here.