Tripped by Bradford Assay

Bradford assay is a commonly used method to quantify (estimate) protein levels. Unfortunately, I was confused by the protocols. I have done the assay before. However, different people appear to use different protocols. I do not know which is right (more accurate). Seniors recommend that I read the manufacturer's protocol which should be optimized (Why did not I think of that?!!). Well, it turns out that everyone is partially right in some parts and partially "wrong" (perhaps they took into consideration certain factors which I did not).

Ok. So after consulting the manufacturer's protocol and verifying it with Current Protocols in Molecular Biology and a certain university's protocol, I have come to a final protocol.

1. Protein standards will range from 0.05mg/ml to 0.5mg/ml BSA (0, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50)
2. Add 10ul of standard/sample into each well.

3. Add 200ul of 5x diluted Bradford regent.

4. Incubate for at least 5 minutes.

5. Read at 595nm.

6. Perform standard curve every time I do the assay (to account for different incubation time).

1st Month Review fo Project Final Fantasy

One month flashed past very very quickly. It has been one month since Project Final Fantasy started. Here is a list of what has been done:

1. 
   
   Procurement of materials is mostly done.
2. 
   
   Assay for target organism appears to be going well.
3. 
   
   Additional measures have been put in place to prevent contamination.
4. 
   
   A modest stock of target organism has been propagated (17 from true breed and 17 from cross breed).
5. 
   
   Primary drug treatment in progress.

The main concern I have at the moment is that the progress is relatively slow. I fear not being able to complete all the tasks in time. My speed in performing tasks are also relatively slow -- This could pose a problem when the equipment are more frequently utilized by others. I am also behind time on reading.

Why the Lack of Progress in Reaping Benefits from Human Genome Sequence

An article from yesterday's newspaper commented on the progress (or the lack of progress) in reaping the rewards of having sequenced the human genome. According to the article, although the sequencing (determination of the order of As, Ts, Cs and Gs of your genome) of human genome was completed 10 years ago and scientists have since further their understanding on human diseases such as diabetes and cancers. However, this has yet to translate to clinical applications, with the most advanced therapies under still under clinical trials.

Perhaps it was over optimism or lack of sufficient knowledge in those days (inevitable because science is always progressing), the people involved in the sequencing projects were hawking the potential of having the genome sequences: cancer therapies that targets precisely the genomic abnormalities and treatment of other genetic diseases such as osteoporosis and autoimmune diseases such as lupus.

Obtaining the human genome sequence is, no doubt, a big achievement.  Knowing the sequence will allow us to predict outcome of cancer treatments (genetic markers as prognostic factor) for example (if someone has a particular mutation in a gene or multiple mutations in multiple genes, what how good will be his response to anti-cancer therapy). Having your genomic sequence will also allow detection of diseases caused by the absence of certain genes or by having too many copies of certain genes.

However, knowing having the genome sequence is merely the start of a long journey. Having a particular gene that codes for a disease does not mean you will have the disease. A disease can involve single gene or multiple genes. If it is a single gene, then dominant and recessive alleles will come into play when determining if you will have the disease. Humans have two copies of a gene in their genome (in general). If one of the copy is "defective" and this copy is dominant, then having one single "defective" copy means you will have the disease (if we ignore other pathways that can compensate for this defect). However, if the "defective" copy is recessive, then you will need two of such "defective" copies to have the disease -- you are safe as long as you have a normal copy which is dominant. In this regards, the genome sequence will tell you that you have a defective copy but not whether it is recessive or dominant (which will require genetic studies to elucidate). Therefore, having a "defective" copy of a gene does not mean you will the disease.

Furthermore, for the effect of the "defective" gene to manifest, it has to be expressed. Not all genes are expressed at all times. Some are expressed at specific points of time (such as during fetal development or during puberty) or under specific conditions (insulin is expressed when there is high blood glucose level). Therefore, even if your genome sequence shows that you have a "defective" gene, you may not have the disease if the gene is not expressed. In addition, there is the epigenetics factor to consider. Modification of proteins bound to your DNA may alter the accessibility to factors needed to express the gene. Therefore, again, the correlation between having a gene and disease is not so straightforward.

The problem of correlating a gene and a disease becomes much more complex when it is a multiple gene system. A mutation of one or more genes may alter the system and the interactions between these genes may or may not result in you having the disease. Treatment targeting a "defective" gene may perturb these interactions, resulting in exacerbating the disease or improving the patient's conditions. Having the genome sequence does not tell you which gene interacts with which gene(s) or even what are the function(s) of each individual gene.

To put it simply, sequencing the genome is merely analogous to deciphering the spelling of each word. However, we do not know what the word means; we do not know what it meaning will be conveyed when we put several words together into a sentence. A lot more work, a lot more time, a lot money is needed if we are to realize the potential of human genome sequence.

Contaimination! Contamination! Contamination!

Contamination in 2 flasks and 2 wells!!! Contamination!!! Unforgivable! I think contamination reflects that I am not a very careful person. Ok. Some of the senior students also get contamination now and then (relatively infrequent though.) Very problematic. I did not experience contamination in the first year and now, I get contamination in 3.5 weeks! I have take a more extreme aseptic measures. This is very troublesome, especially since I have to boost my speed of doing things. Looks like ethanol consumption will rise by quite a bit in the coming days.

Project Final Fantasy Update 3

It has been 3.5 weeks. What have I done? Well... not a lot. Not the actual work anyway. They will begin next week. Hopefully. Must work harder while morale is higher. My morale is always higher when things begin. There is apprehensive of course. However, future is always brighter when I am dreaming of the potential of this work.

After a few times of problems with plating the proper number of cells (various people suggested various methods), I think I finally got things right. Finally. Some do consider individual cells of multicellular organisms as an organism. I do not. Simply because... well... they are not complete organisms. Besides, if you consider them complete organisms, imagine carrying out "massacre" of tens of millions of cells every day.

In this 3.5th week of Project Final Fantasy, I had three dinners with friends. Just this week, there was the dinner with science research people (Too bad our doctors did not attend) and dinner with secondary school gang. Everyone seems to be doing well relatively speaking. These outings are the calm in this turbulent period of "Battle". I really and acutely felt the calm during science research people outing, while we are around the Helix Bridge area. If I remember, the last time I had a similar feeling was during secondary school CCA chalet.

Truthfully, I am not one to root for prolonged period of calmness in work. Calmness is good. Stability, predictability and a sense of feeling good. Especially if you have already found your significant Other. I think I like challenges. I enjoy competition. It raises my game. I think inevitably, when you mix with strong (or stronger) people, you become stronger over time as well. Also, the kind of euphoria when the dread of failing is banished with unexpectedly good outcome is unbelievable (The lack of people to share this euphoria can be a downer though). Yup. I think the thirst for competition, the craving for euphoria of success and the burning desire to beat a challenge are driving me onwards (and perhaps further away from romance though).

Then again, I think I have become a better loser. More willing to accept responsibility for making mistake, admit mistake and face mistakes. This is essential I think. If I do not face mistakes, then given my highly competitive nature, I will (in the words of a senior) "crash and burn".

Successful Stage 6

In the fight with 8 stages, I supposed I can claim to win Stage 1,2,3,4. Stage 5 was kind of a draw or even a loss (though most people would claim otherwise) - 2 of enemies are tough to beat and I got sucker punched by 2 others. The main problem then was the lack of motivation. Or perhaps winning the previous stages had made me over-confident of my abilities. Very, very big mistake. The good thing is, they reminded me of my weaknesses.

As much as I hate to admit these weaknesses, I do know that if I do not admit their existence, I will never improve. It is better to face up to reality and challenges than run away from them or bury my head in the ground. I did some reflection and tried to put new measures in place for Stage 6... and Stage 6 is another success. All right, the success of Stage 6 belonged not only to me, but to my mentor and seniors. They played a very big part in this. I can never thank them enough. Now, as I move on to Stage 7 and 8, bigger challenges await. To achieve true victory, I have to overcome these.

So, as you can see, I am someone who likes to "fight". Overly competitive they say. Then again, this is how I live most of the time. "Fighting" is the only way of life that I know. Perhaps this is why I am incapable of proper love.

Watch What Your Kids Put into Their Mouths

Being interested in infectious diseases, I was a little shocked to see something today. At a restaurant, a small kid (3-4 years old by my reckoning) was playing around. At one point, he used his index finger to rub the floor (The restaurant staff was mopping the floor. Perhaps the kid was curious about the shiny and slippery surface). The Mother (I presume), carried him back to her seat. The aunt (another assumption) gave the kid a french fry. Not into the mouth of the kid, but letting him hold it by the very hand that touched the floor! You would think that with the recently outbreak of hand-foot-mouth disease, parents and guardians would be more careful.

Now. Infectious disease are scary. However, most of the time, they can be easily prevented! Simple things like avoid spitting, covering up mouth or nose when you cough or sneeze, remove stagnant water and wash your hands before eating can go a long way in keep you healthy. Alright. I hear some of you saying that what if you did your part, but others do not. I have seen people (mostly older generations, though these are only anecdotal evidence) spitting; people coughing without covering their mouth etc. I think we need more education. Things may look alright now. However, imagine if there is a influenza pandemic (Experts believe it is imminent. The only question is when it will happen.) or an outbreak of multi-drug resistant tuberculosis. Then what? Do we need really need a major event like SARS to wake us up?

Yes. People have complained about being a nanny state. The question is, how can we not be a nanny state when we cannot do things right ourselves?