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.